172 Research Article

IntroductionAxon growth is regulated by numerous extracellular cues,both soluble and bound, and the growth cone is thought tointegrate this information (Tessier-Lavigne and Goodman,1996; Goldberg et al., 2002; Huber et al., 2003). For example,components of the extracellular matrix (ECM) potentiateaxon outgrowth of CNS neurons if growth factor signals arepresent (Edgar et al., 1988; Goldberg et al., 2002; Liu et al.,2002).

Many receptors for soluble factors and ECM moleculeshave been identified in the growth cone. Other putativereceptors have been observed, but their functions areunknown. We have described a highly heterogeneousglycoprotein, gp93, that is enriched in growth conemembranes (Quiroga and Pfenninger, 1994) and identified itas ‘signal regulatory protein’ (SIRP�), also known as SHP(Src homology domain 2-containing phosphatase) substrate-1(SHPS-1), BIT or P84 (Fujioka et al., 1996; Comu et al., 1997;Kharitonenkov et al., 1997; Sano et al., 1997; Wang et al.,2003). SIRP� is a heavily glycosylated, Ig superfamilytransmembrane protein that contains three extracellular Igdomains and two intracellular ITIM (immunoreceptortyrosine-based inhibitory motif) sequences. Phosphorylationof the ITIM tyrosines triggers binding and activation of thetyrosine phosphatases, SHP-1 and SHP-2 (Fujioka et al., 1996;Kharitonenkov et al., 1997). CD47, or integrin-associatedprotein (IAP) is believed to be a ligand of SIRP� (Jiang et al.,1999; Seiffert et al., 1999). SIRP� is expressed throughout thecentral nervous system (Comu et al., 1997; Mi et al., 2000),but in growth cones from different developing brain regions it

exhibits differential glycosylation (Li et al., 1992; Wang et al.,2003; see also, van den Nieuwenhof et al., 2001). Thefunctional significance of this neuron-type-specificglycosylation is unknown, but it might alter the binding ofSIRP� to CD47 or other, unknown ligands (Ogura et al.,2004).

Depending on the types of ligand and cell involved, SIRP�binding to CD47, SHP-1 or SHP-2 positively or negativelyregulates a variety of cellular functions, includingmitogenesis, motility and adhesion (Cant and Ullrich, 2001;Oshima et al., 2002). There are data to suggest that, inneurons, SIRP� may be involved in the regulation of survival,neurite outgrowth and/or synapse formation and maintenance(Chuang and Lagenaur, 1990; Comu et al., 1997; Sano etal., 1997; Jiang et al., 1999; Araki et al., 2000; Mi et al.,2000).

As shown here, SIRP� is located, in part, in lipidmicrodomains (LMDs). Such LMDs (Simons and Ikonen,1997; Brown and London, 1998; Simons and Toomre, 2000)seem to act as platforms for signal transduction initiated byseveral neurotrophic factors, and they are important forneuronal cell adhesion and axon guidance (Saarma, 2001; Tsui-Pierchala et al., 2002; Guirland et al., 2004). In order todetermine the function of SIRP� in the growth cone, weexamined (1) its spatial distribution; (2) its phosphorylation andSHP-2 binding in response to the ECM molecule laminin, andto growth factors; and (3) the effects of overexpressing SIRP�cytoplasmic fragment. The results indicate that SIRP�modulates axonal growth when growth cone integrins areactivated by laminin.

The ‘signal regulatory protein’ SIRP�� is an Ig superfamily,transmembrane glycoprotein with a pair of cytoplasmicdomains that can bind the phosphatase SHP-2 whenphosphorylated on tyrosine. SIRP�� is prominent in growthcones of rat cortical neurons and located, together with thetetraspanin CD81, in the growth cone periphery. SIRP�� isdynamically associated with Triton-X-100-sensitive, butBrij-98-resistant, lipid microdomains, which also containCD81. Challenge of growth cones with the integrin-bindingextracellular-matrix (ECM) protein, laminin, or with thegrowth factors, IGF-1 or BDNF, increases SIRP��phosphorylation and SHP-2 binding rapidly andtransiently, via Src family kinase activation;phosphorylated SIRP�� dissociates from the lipidmicrodomains. A cytoplasmic tail fragment of SIRP��

(cSIRP��), when expressed in primary cortical neurons, alsois phosphorylated and binds SHP-2. Expression of wild-type cSIRP��, but not of a phosphorylation-deficientmutant, substantially decreases IGF-1-stimulated axonalgrowth on laminin. On poly-D-lysine and in controlconditions, axonal growth is slower than on laminin, butthere is no further reduction in growth rate induced by theexpression of cSIRP��. Thus, the effect of cSIRP�� on axongrowth is dependent upon integrin activation by laminin.These results suggest that SIRP�� functions in themodulation of axonal growth by ECM molecules, such aslaminin.

Key words: Growth cone, SIRP�/SHPS-1, Growth factors, Laminin,Src family kinases, Growth control

Summary

Functional analysis of SIRP�� in the growth coneXiaoxin X. Wang and Karl H. Pfenninger*Department of Cell and Developmental Biology, University of Colorado School of Medicine and University of Colorado Cancer Center, Aurora,CO 80010, USA*Author for correspondence (e-mail: Karl.Pfenninger@uchsc.edu)

Accepted 27 September 2005Journal of Cell Science 119, 172-183 Published by The Company of Biologists 2006doi:10.1242/jcs.02710

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ResultsImmunolocalization of SIRP� in cultured growth conesThe distribution of SIRP� in intact growth cones was studiedby indirect immunofluorescence in cultures of primary ratcortical neurons (Fig. 1). Since the antibody did not stain intactneurons (data not shown), a mild detergent (1% Brij 98) wasused to permeabilize membranes in order to preserve LMDs.Specific fluorescence was observed throughout the neuron (notshown). As is the case for other membrane components intransit to the growth cone, a high level of SIRP� as seen in theaxon. In growth cones cultured on laminin (Fig. 1A-D) SIRP�staining was evident as variously sized spots that wereparticularly prominent in the periphery. The staining extendedinto the finest filopodia and their tips, and it followed the actinbundles identified by phalloidin labeling. This labeling patternwas independent of the substratum used for culture. On poly-D-lysine, which is not a physiological substrate and cannottrigger integrin signaling, cortical neurons had larger growthcones bearing more abundant but shorter filopodia (Fig. 1E,F).Despite the difference in growth cone morphology, SIRP�distribution in the growth cone was again punctate andprimarily peripheral, in the filopodia.

We also compared the distribution of growth cone SIRP� tothat of an LMD-resident tetraspanin, CD81 (Hemler, 2003), by

double immunofluorescence. As seen in Fig. 1G-I, CD81 wasdistributed in discrete puncta, primarily in distal regions of thegrowth cone (grown on laminin). Many of these punctaoverlapped with SIRP� immunoreactivity, particularly alongthe filopodia and at their tips. These data indicate substantialcolocalization of SIRP� with CD81 in growth cones.

Distribution of SIRP� in growth cone LMDsThe punctate co-distribution of CD81 immunoreactivity withSIRP� in growth cone plasmalemma may indicate itscompartmentalization in LMDs. Therefore, we investigated ifgrowth cone SIRP� co-fractionated with LMDs.

Three criteria are typically used to define LMDs: (1)insolubility in non-ionic detergents; (2) flotation in a sucrosedensity gradient; and (3) detergent solubility in the presence ofcholesterol-sequestering agents (Simons and Toomre, 2000).Cold TX100 is used most commonly for identification of‘rafts’, microdomains enriched in cholesterol,glycosphingolipid and GPI-anchored proteins. Using thismethod, cold TX100-resistant membranes were prepared fromgrowth cone particles (GCPs) and isolated by flotation in low-density fractions recovered from a bottom-loaded,discontinuous sucrose gradient. Fractions were analyzed byimmunoblotting as shown in Fig. 2A. Src, a known resident

Fig. 1. (A-F)Immunolocalization of SIRP� ingrowth cones of cortical neuronscultured either on laminin (A-D)or poly-D-lysine (E,F). Bar, 10�m. (G-I) Double-immunofluorescence of SIRP�and CD81 in a growth cone onlaminin. Bar, 5 �m. Growthcones were double-labeled withanti-SIRP� antibody (AlexaFluor® 594-conjugatedsecondary antibody;B,D,E,F,G,I) and FITC-phalloidin (C,D,F) or anti-CD81(Alexa Fluor® 488-conjugatedsecondary antibody; H,I). D isthe merged image of B and C(overlap appears yellow), and Ais the corresponding phase-contrast image. F is the mergedimage of E with that ofphalloidin labeling (not shownseparately). I is the mergedimage of G and H and showsextensive co-localization(yellow) of SIRP� and CD81.Images are 0.1 �m opticalsections obtained by digitaldeconvolution. The arrows in Dpoint to SIRP�immunoreactivity in filopodialtips. The arrows in I indicatefilopodial tips with prominentSIRP� and CD81 overlap.

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protein of lipid rafts, was mainly recovered in low-densityfractions (0.6-0.7 M), as expected. However, we did notobserve partitioning of SIRP� into these low-density fractions.

More recently, LMDs have been described that exhibitdifferent detergent solubility characteristics (Chamberlain,2004). For example, tetraspanin-containing LMDs can bemaintained only in the presence of milder detergents, such asBrij 97 and Brij 98, but not TX100. Brij 97 is less harsh thanTX100, and Brij 98 is even milder. In Brij 97, we found nopartitioning of SIRP� or of the tetraspanin, CD81, into low-density fractions of sucrose gradients (Fig. 2B). However, asignificant proportion of SIRP�, as well as CD81 and Src, wererecovered from low-density fractions (0.3-0.7 M) in Brij 98(Fig. 2C). In the same preparation, the transferrin receptor, aprotein not associated with LMDs (Harder et al., 1998), wascompletely solubilized and recovered from the high-densityfractions. This suggests that SIRP� significantly associateswith Brij 98-resistant LMDs.

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To test whether SIRP� is associated with LMDs in acholesterol-dependent manner, GCPs were pre-treated withsaponin or M�CD to disrupt LMDs by cholesterol perturbation(Rothberg et al., 1990; Cerneus et al., 1993; Ilangumaran andHoessli, 1998; Ledesma et al., 1998; Kim and Pfeiffer, 1999).As seen in Fig. 2D,E, saponin or M�CD pre-treatmentdramatically reduced the partitioning of SIRP� and Src intolow-density fractions. These results indicate that SIRP� co-localizes with CD81 in TX100-sensitive but Brij 98-resistantLMDs.

Transient phosphorylation of growth cone SIRP� inresponse to laminin, IGF-1 and BDNFPotent extracellular signals for growing neurons are theneurotrophic factors, BDNF and IGF-1, which operate throughreceptor tyrosine kinases, and ECM molecules, such aslaminin, which activate tyrosine kinases via integrins. Whilesuch factors are known to stimulate SIRP� tyrosylphosphorylation in other cells (Ohnishi et al., 1999; Maile andClemmons, 2002a), this is not known for the growth cone.Therefore, we proceeded to examine SIRP� responses to suchexogenous signals in GCPs, a preparation highly enriched inre-sealed, primarily axonal growth cones (Pfenninger et al.,1983; Lohse et al., 1996). Previous studies have shown thatBDNF and IGF-1 stimulate axonal growth, and that GCPscontain functional receptors for them (Davies et al., 1986;Aizenman and De Vellis, 1987; Pfenninger et al., 2003).Therefore, we chose IGF-1 and BDNF to test the effects ofgrowth factors on SIRP� in GCPs. As an ECM molecule, weselected laminin for these experiments because it is a preferredgrowth substrate for developing CNS neurons.

Because we used laminin in suspension rather thanimmobilized on a solid phase for studying the short-termresponses of the GCPs, we first tested whether soluble laminincould indeed stimulate integrin signaling. Plating GCPs onlaminin, i.e. laminin-induced integrin engagement, activatesSrc and increases Src binding to the cytoskeleton (Helmke etal., 1998). Therefore, we measured these parameters insuspended GCPs treated with soluble laminin. As shown inFig. 3A with anti-Src-pY418 (which recognizes only activatedSrc), laminin treatment for 1 minute at 37°C increasedactivated Src in GCPs by about 60% compared to controlincubation (an antibody to the p85 subunit of PI 3-kinase wasused to show equal loading). We also probed the cytoskeleton

Fig. 2. Distribution of SIRP� in lipid microdomains. Growth coneparticles (GCPs) were treated with different detergents (indicated onthe left of each panel) and fractionated in sucrose density gradients(A-E); the sucrose concentrations of the fractions are given at the topof each lane. Fractions were collected from top to bottom of thegradients. Fractions were analyzed by western blot with the indicatedantibodies (IB, right; �=anti). (C) The experiment without detergentserves as a control for non-solubilizing conditions. Anti-transferrinreceptor (TfR) blot is a control for Brij 98, showing thattransmembrane proteins such as TfR are solubilized under theseconditions. (D) Saponin was added before the Brij 98 extraction ofGCPs to disrupt cholesterol interactions. The controls withoutsaponin treatment produced the same results as in C and, therefore,are not shown again. (E) M�CD was used to extract cholesterol. Inthese experiments, samples of different density (range indicated onthe top) were pooled to represent high-, middle- and low-densityfractions. For detailed description, see Results.

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fractions prepared from GCPs with anti-Src antibody to revealassociated total Src protein. Laminin treatment for 5 minutesat 37°C increased Src association with the cytoskeletonfraction by about 70% compared to control (Fig. 3B; in thisexperiment, each lane was loaded with the total cytoskeletonfraction prepared from equal GCP samples). Interestingly,SIRP� was not detectable in these cytoskeletal preparations,but SHP-2 was clearly present. The observed activation andincreased cytoskeletal association of Src indicated that lamininapplied to GCPs in suspension activated Src (see Helmke et al.,1998).

To determine whether laminin, BDNF or IGF-1 stimulationchanged tyrosine phosphorylation of SIRP�, GCPs wereincubated with the different factors for various times at 37°C,and SIRP� was immunoprecipitated. The immunoprecipitateswere resolved by SDS-PAGE, blotted, and blots probed withanti-pTyr. In addition, blots were probed with anti-SHP-2 toassess whether SHP-2 co-immunoprecipitated with SIRP�(SHP-1 was not considered because it is a hematopoietic-cellphosphatase, whereas SHP-2 is ubiquitously expressed) (Neelet al., 2003). As shown in Fig. 4A, all three factors inducedrapid phosphorylation of SIRP�. The increases over non-treated controls were 37±5% for laminin, 39±4% for IGF-1,and 21±4% for BDNF (means±s.e.m.; n�4) after 1 minute at37°C. These increases were modest but statistically significant(P�0.01). It is known that GCPs exhibit high levels of proteinkinase activity in control conditions (Helmke et al., 1998) sothat, even without stimulation, SIRP� phosphorylationcontinued to increase over the 5-minute observation period(Fig. 4B). However, this increase was reduced for GCPs treatedwith laminin, IGF-1 or BDNF. As a result, we observeddecreases relative to control levels after 5 minutes at 37°C.These decreases were 25±4% for laminin, 20±5% for IGF-1,

and 23±4% for BDNF (means±s.e.m.; n� 4). Again, this wasstatistically significant (P�0.01). Thus, the stimulation ofSIRP� phosphorylation by the three factors was transient.Simultaneously with the increase in phosphorylation, the threefactors enhanced co-immunoprecipitation of SHP-2 after 1minute at 37°C (Fig. 4A). Likewise, we observed a decrease inSHP-2 relative to control after 5 minutes at 37°C (Fig. 4B).The changes paralleled those of SIRP� phosphorylation andalso were statistically significant. These results indicated thatlaminin, BDNF and IGF-1 stimulated tyrosyl phosphorylationof SIRP� and its association with SHP-2 in isolated growthcones, and that these changes were rapid and transient. We alsotested combinations of factors in such experiments. Lamininplus IGF-1 or laminin plus BDNF did not significantly increasetyrosyl phosphorylation or SHP-2 binding of SIRP� above thelevels obtained with laminin, IGF-1 or BDNF alone (data notshown). Thus, the effects of laminin and growth factors did notseem to be additive or synergistic under the experimentalconditions used.

The stimulation of SIRP� phosphorylation by laminin,BDNF and IGF-1 raised the question of which kinase(s) wereresponsible for this effect in growth cones. It has been reportedthat Src-like kinases may phosphorylate SIRP� in fibroblastsupon integrin-mediated adhesion to fibronectin (Oh et al.,1999). To determine whether Src-like kinases were indeedresponsible for SIRP� phosphorylation in growth cones, weexamined the effects of the selective Src kinase inhibitor, PP2,on laminin-, IGF-1- or BDNF-induced SIRP� phosphorylationand SHP-2 association. The control was GCPs withoutstimulation. Incubation of GCPs with 1 �M PP2 reducedlaminin-, IGF-1-, or BDNF-stimulated SIRP� phosphorylation(Fig. 4C). As in the other experiments (Fig. 4A), SHP-2 co-immunoprecipitation paralleled phosphorylation, i.e. PP2decreased SHP-2 association with SIRP� (Fig. 4D). Theseblots were probed with anti-SIRP� as a loading control tonormalize SIRP� phosphorylation and co-immunoprecipitation of SHP-2. Stimulation with these factorsnormally increased SIRP� phosphorylation and SHP-2 bindingto SIRP� by 20-40% relative to control at 1 minute (see Fig.4A). However, PP2 greatly reduced SIRP� phosphorylationand SHP-2 binding from 100 (normalized arbitrary units,control without PP2) to about 18-21% (n=2) and 13-17%(n=3), respectively, for all stimulation conditions. Thisreduction was highly significant (P<0.01). In the absence ofstimulation, SIRP� phosphorylation and SHP-2 binding alsowere reduced after PP2 treatment, to a level that was notstatistically distinguishable from that of the stimulated, PP2-treated samples. Unlike PP2, PP3, a negative control for PP2,did not reduce SIRP� phosphorylation (Fig. 4C). The dataindicated that the increase in SIRP� phosphorylation and SHP-2 association stimulated by laminin, IGF-1 or BDNF in growthcones depended on Src-like kinase(s). However, these resultsdid not completely exclude the possible participation ofanother SIRP� kinase, since PP2 treatment did not fullyabolish SIRP� phosphorylation and SHP-2 association.

Phosphorylation shifts SIRPa out of LMDsThe results described above suggested that at least a sizablefraction of SIRP� is located in LMDs, and that growth coneSIRP� may be involved in the processing of ECM andgrowth factor signals. Next, we investigated how the

Fig. 3. The addition of laminin to GCPs in suspension activates Srckinase. (A) Western blot of laminin-treated or control GCPs, probedwith the indicated antibodies (IB; �=anti). p85 serves as a loadingcontrol. Numbers below the blot are the average ratios for phospho-Src over p85 (arbitrary units normalized to control ± s.e.m.; n=2).(B) Cytoskeleton fractions prepared from GCPs with or without priorlaminin treatment (each lane was loaded with the total cytoskeletalpreparation from equal GCP samples). Samples were subjected towestern blot with the indicated antibodies (IB). The numbers belowthe blot represent quantitation of Src (arbitrary units normalized tocontrol ± s.e.m.; n=2). Note that SIRP� was not detected in thecytoskeleton fraction, but that SHP-2 was present.

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compartmentalization in LMDs related to the phosphorylationof SIRP�.

We used three different conditions to change thephosphorylation state of SIRP�, and assessed its distributionin lipid microdomains. The Src kinase inhibitor, PP2, served

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to block SIRP� phosphorylation. Laminin was used tostimulate SIRP� phosphorylation, and vanadate, a broad-spectrum tyrosine phosphatase inhibitor, caused hyper-phosphorylation of SIRP�. Following treatment with one ofthese reagents, GCPs were solubilized in Brij 98 and

Fig. 4. Laminin, IGF-1 and BDNF stimulate transient phosphorylation of SIRP� and its association with SHP-2 in GCPs. (A) GCPs with orwithout stimulation were immunoprecipitated with anti-SIRP� antibody after 1 minute of incubation. Immunoprecipitates were analyzed bywestern blot with anti-pTyr and anti-SHP-2 to examine SIRP� phosphorylation and SHP-2 co-immunoprecipitation, respectively (�=anti). Thebar graphs show quantitation of SIRP� phosphorylation (left) and of SHP-2 co-immunoprecipitation (right), expressed as the percentagechange over control level. Results are means ± s.e.m. of at least four independent experiments. *P�0.01 compared to control. (B) The sameexperiment as in A, but 5 minutes incubation time. The bar graphs show decreases in phosphorylation and SHP-2 co-immunoprecipitationrelative to control. (C,D) Effects of Src family kinase inhibitor on laminin-, IGF-1- and BDNF-stimulated tyrosyl phosphorylation (C) andSHP-2 binding of SIRP� (D). The Src kinase inhibitor, PP2, was added to GCPs prior to incubation in control or stimulated conditions. PP3added in control conditions served as a negative control for PP2. After 1 minute incubation, GCPs were solubilized and SIRP�immunoprecipitated. Western blots of SIRP� immunoprecipitates were probed with anti-pTyr to examine SIRP� phosphorylation (C) or withanti-SHP-2 to reveal SHP-2 co-immunoprecipitation (D). In addition, they were probed with anti-SIRP� to control for SIRP� loading. Notethat PP2, even with stimulation, reduced SIRP� phosphorylation and co-immunoprecipitation of SHP-2 to below control levels without PP2treatment (C and D) or with PP3 treatment (C). The numbers below the blots are mean ratios ± s.e.m. (n=2 in C; n=3 in D) of pTyr or SHP-2label over SIRP� label (arbitrary units, normalized to control without PP2 treatment).

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fractionated in density gradients containing five steps(0.5/0.7/0.9/1.0/1.33 M sucrose). The distribution of SIRP�and Src in the gradients was determined by western blotanalysis. In the presence of PP2, virtually all SIRP� wasrecovered in the LMD fractions (this was normalized to 100%).Laminin and vanadate treatment, however, reduced thepercentage of SIRP� in low-density fractions to only 27% and6%, respectively, of the level found after PP2 treatment (Fig.5). This indicated for SIRP� an inverse relationship betweenthe phosphorylation state and co-fractionation with lipidmicrodomains. Src, by contrast, remained largely (62-70%) inthe low-density fraction, even after PP2 treatment.

The cytoplasmic tail of SIRP� expressed in culturedneurons is phosphorylated and binds SHP-2In order to study SIRP� function in the growth cone we wantedto perform misexpression experiments. The association ofSIRP� with LMDs and the fact that expression of itscytoplasmic tail (cSIRP�) has a dominant-interfering effect onTNF� signaling (Neznanov et al., 2003) prompted us to choosethe soluble cSIRP� fragment for these experiments.Overexpression of cSIRP� in the neuron may have a dominant-interfering effect on growth cone function because it maycompete with wild-type SIRP� for phosphorylation of theITIM motif and for SHP-2 binding. Therefore, we first assessedcSIRP� phosphorylation and SHP-2 binding in transfectedcortical neurons. After 48 hours in culture, neurons transfected

with the fusion construct GFP-cSIRP� were harvested andsolubilized in the presence or absence of ATP plus vanadate(to inhibit protein tyrosine phosphatases). Weimmunoprecipitated with anti-GFP antibody and probedwestern blots of the immunoprecipitates with (i) anti-GFP(loading control), (ii) anti-phosphotyrosine and (iii) anti-SHP-2 antibodies. As shown in Fig. 6, anti-GFP precipitated a GFP-positive fusion protein of the appropriate molecular mass (45kDa), and the intensity of the band in the two experiments wasvery similar. In phosphorylating conditions, anti-GFPrecognized a second band with a slightly higher molecularmass, and this band was pTyr-positive. In the absence of ATPand vanadate, anti-pTyr and anti-SHP-2 demonstrated very lowlevels of phosphorylation and SHP-2 binding. These weregreatly increased in phosphorylation conditions. For twoindependent experiments, the average increase was tenfold andfivefold for tyrosine phosphorylation and SHP-2 binding,respectively. Thus, cSIRP� expressed in cultured corticalneurons was phosphorylated and bound SHP-2.

SIRP� regulates the rate of axonal outgrowthOur biochemical experiments showed that laminin and theaxonal growth factors IGF-1 and BDNF stimulated SIRP�phosphorylation and the assembly of SIRP�-SHP-2 complexes

Fig. 5. Phosphorylation shifts SIRP� from lipid microdomains(LMDs). The bar charts show the percentage of SIRP� (upper) and,for comparison, of Src (lower) recovered in LMDs under differentexperimental conditions. Data were normalized to the value in PP2-treated GCPs. The values are expressed as means ± s.e.m. for threeindependent experiments.

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Fig. 6. cSIRP� expressed in neurons is phosphorylated and bindsSHP-2. Cortical neurons transfected with GFP-cSIRP� were culturedfor 48 hours. Cells and neurites were harvested, permeabilized with�-escin and incubated for 10 minutes at 37°C in the presence orabsence of ATP and vanadate. GFP-cSIRP� was immunoprecipitatedfrom a TX100 extract of the samples and analyzed by western blot(IB; �=anti). Probing with anti-pTyr antibody (Cy5-conjugatedsecondary antibody) revealed greatly increased phosphorylation of asingle band in the ATP plus vanadate-treated samples (top panel).The blot was stripped, checked for absence of remaining Cy5 signal,and re-probed with anti-GFP (middle panel). This confirmed (i)presence of approximately equal amounts of GFP-cSIRP� in bothsamples, and (ii) the identity of the pTyr-positive band. The sameblot was also probed with anti-SHP-2 (Cy3-conjugated secondaryantibody), revealing SHP-2 co-precipitation in ATP plus vanadatesamples only. Representative image of two independent experimentsare shown.

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at the growth cone plasma membrane. This suggested thatSIRP� might play a functionally important role in theregulation of axonal growth. To test this possibility, weoverexpressed cSIRP� in dissociated primary cortical neuronsand studied their outgrowth in culture. Our molecular tools fortransfection were: (i) plasmid pcDNA3.1-cSIRP�, whichencodes a wild-type, soluble cytoplasmic fragment of SIRP�with dominant-negative properties in other systems (Neznanovet al., 2003; Neznanov et al., 2004), and (ii) plasmid pcDNA-cSIRP�-FYFF, which encodes a phosphorylation-deficientversion of the same SIRP� cytoplasmic fragment, with threeof the four ITIM tyrosines changed to phenylalanines. Theexpression of the two proteins, cSIRP� and cSIRP�-FYFF,was confirmed by immunoblot with anti-SIRP� (data notshown). To enable the identification of transfected neurons, weco-transfected with GFP. To establish positive correlation ofGFP fluorescence with cSIRP� and cSIRP�-FYFF expression,we used an anti-SIRP� antibody raised against the C-terminalpeptide (anti-cSIRP�) that bound only at a very low level tonon-transfected neurons (because SIRP� was present inlimited amounts and/or because of steric hindrance). Inneurons overexpressing cSIRP� or cSIRP�-FYFF, however,this antibody produced strong signals. Fig. 7A-C showsneurons co-transfected with GFP and cSIRP� plasmids. Wefound that all neurons expressing GFP also exhibited stronganti-cSIRP� reactivity. Non-transfected neurons were notdetectable either by GFP or anti-cSIRP� fluorescence. Thesame results were obtained with GFP and cSIRP�-FYFF co-transfections (data not shown). This confirmed that GFP-positive neurons also expressed cSIRP� or cSIRP�-FYFF.

To define the phenotype induced by overexpression ofcSIRP�, we first examined whether growth cone morphologywas changed. Fig. 7D-F shows typical, highly motile growth

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cones from cortical neurons on laminin. As seen in theserepresentative images, the growth cone area, the approximatenumbers, lengths, and positions of filopodia, and othermorphological features did not appear to be affected bytransfection with GFP only (Fig. 7D), with cSIRP� plus GFP(Fig. 7E), or with cSIRP�-FYFF plus GFP (Fig. 7F). Thisresult also indicated that the overexpression of cSIRP� orcSIRP�-FYFF was not neurotoxic under the experimentalconditions used. Interestingly, in the growth cones transfectedwith cSIRP� plus GFP, the immunoreactivity of cSIRP�(labeled with an antibody to a C terminal peptide, anti-cSIRP�)was not diffusely distributed as expected from its solubility.Instead, as shown in Fig. 7H (compare with Fig. 7I), cSIRP�was enriched in the thinner periphery of the growth cone,creating an image reminiscent of the distribution of membrane-anchored SIRP�. All the more surprisingly, perhaps, thedistribution of endogenous SIRP� (selectively labeled with anantibody to the external domain) was not affected (Fig. 7G).

Next we analyzed axonal growth rates in control versuscSIRP�-expressing neurons, in defined culture mediumcontaining 10 nM IGF-1. Growth cone advancement involvesrepeated cycles of extension and stalling, sometimes eveninvolving growth cone collapse or retraction. To average outthese short-term dynamic variations, we measured the growthrate as the displacement of the growth cone over periods of 2-3 hours and expressed it in �m/hour. Fig. 8A shows two axonsgrowing on laminin, one transfected with GFP only (control),and another transfected with both cSIRP� and GFP (Fig. 8A,fluorescent images on top). The phase-contrast images showthe same axons immediately after the addition of 10 nM IGF-1 to the culture and 1 hour later. The distance of growth coneadvancement is indicated by two arrows. The control growthcone (left panels) advanced much further than the cSIRP�-

transfected growth cone (right panels). Bothmaintained their lamellipodial and filopodialdynamics during the observation period, however.When a population of such axons was analyzed (Fig.8B) quantitative analysis revealed that cSIRP�overexpression (n=14) reduced the growth rate to33% of the control value. This change was significantstatistically (P<0.05). However, overexpression of

Fig. 7. (A-C) GFP-positive neurons express cSIRP� inco-transfected cultures. (A) GFP fluorescence of neurons;(B) the same neurons labeled with anti-cSIRP�, an anti-SIRP� raised against C-terminal peptide (Alexa Fluor®

594-conjugated secondary antibody); (C) merger of thetwo images. Anti-cSIRP� labels non-transfected neurons(abundant in the field) only very weakly so that they arenot visible. Bar, 20 �m. (D-I) Overexpression of mutantSIRP� in the growth cone. (D-F) Representative phase-contrast images of growth cones transfected with: GFPonly (D), cSIRP� plus GFP (E), or cSIRP�-FYFF plusGFP (F). (G) Distribution of endogenous SIRP� (labeledwith an antibody to the external domain) in a growth coneoverexpressing cSIRP�. (H) Immunolocalization ofcSIRP� overexpressed in the growth cone (labeled withanti-cSIRP�). (I) The phase-contrast image of the growthcone in H. A process of a non-transfected neuron, visibleonly in phase-contrast, runs across the image. It is notdetectable by immunofluorescence. Secondary antibodieswere Alexa Fluor® 594 conjugated. Bar, 10 �m.

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the non-functional cSIRP�-FYFF (n=8) did not cause astatistically significant change compared to controls. Thesedata indicated that cSIRP� overexpression had a dominant-interfering effect on axonal growth in cortical neurons culturedon laminin and in the presence of IGF-1. Since the differencebetween cSIRP� and cSIRP�-FYFF was the phosphorylationdeficiency of the latter, this indicated that the effect cSIRP�was specific and related to tyrosine phosphorylation of theITIM motif.

Because laminin stimulates SIRP� phosphorylation ingrowth cones we wanted to determine whether the reductionin axonal growth rate by cSIRP� required the presence of thisECM molecule. Therefore, we also measured axonal growthrates of cortical neurons cultured on poly-D-lysine. In contrastto laminin, poly-D-lysine is a non-physiological substrate thatdoes not activate integrin signaling. As shown in Fig. 8B(right), the growth rate of GFP control axons is reduced onpoly-D-lysine relative to laminin (to 58%; P<0.05). However,cSIRP� overexpression (n=7) did not affect this growth rate(in the presence of IGF-1). There was no statisticallysignificant difference between control and cSIRP� growthrates on poly-D-lysine. These results indicate that thedominant-interfering effect of cSIRP� on IGF-1-stimulatedaxon outgrowth requires laminin-activated integrin signalingin the growth cone.

DiscussionIn our previous study, we identified the highly heterogeneous,prominent growth cone glycoprotein, gp93, as SIRP� (Quirogaet al., 1994; Wang et al., 2003). SIRP� is involved in variouscell functions (Comu et al., 1997; Oshima et al., 2002), but itsmechanism of action in the nerve growth cone has beenunclear.

Localization of SIRP� in growth cones and LMDsIt was known that SIRP� is present in the perikarya, theneurites and the growth cones of cultured neurons (Chuang etal., 1990; Quiroga et al., 1994; Ohnishi et al., 2005). However,its distribution in the growth cone had not been described indetail. After membrane permeabilization with the very milddetergent Brij 98 (rather than the harsher TX100) we observed

a punctate distribution of SIRP�, especially in the distalregions of the growth cone. Sphingolipid/GPI-linked proteinmicrodomains have been shown to coalesce into visibleaggregates by antibody cross-linking in standard protocols(Harder and Simons, 1997; Harder et al., 1998), but not afterglutaraldehyde fixation (Mayor et al., 1994). In the latterconditions, the punctate pattern of SIRP� persisted (data notshown) and, thus, did not appear to be a preparation artifact.SIRP� localization was also consistent with the distributionsreported for �1 integrin and tetraspanins in growth cones (Wuand Goldberg, 1993; Wu et al., 1996; Stipp and Hemler, 2000).Actually, SIRP� co-localized to a substantial degree withCD81 in our experiments, and this was especially evidentalong, and at the tips of, filopodia, again suggesting that SIRP�may be contained in specific LMDs. Our biochemical data doindeed demonstrate that much SIRP� resides in growth coneLMDs, together with CD81.

Various non-ionic detergents have been used to isolateLMDs (Bohuslav et al., 1993; Roper et al., 2000; Bini et al.,2003; Schuck et al., 2003). Brij 98 solubilizes proteins, suchas the transferrin receptor, but preserves the association oftetraspanins with LMDs (Kawakami et al., 2002; Charrin et al.,2003; Hemler, 2003). Like TX100-resistant rafts, Brij 98-resistant LMDs are enriched in cholesterol, sphingolipids andpalmitoylated proteins, but they are depleted of prenylatedproteins (Chamberlain, 2004). We recovered SIRP�, togetherwith CD81, in a Brij 98-resistant, flotable LMD fractionsensitive to cholesterol perturbation and (unlike Src) to TX100at 4°C.

Like tetraspanins, SIRP� is not associated with thecytoskeleton (see Berditchevski and Odintsova, 1999). Thesubstantial co-localization and co-fractionation of SIRP� andCD81 suggest that a large proportion of SIRP� and CD81 mayreside together in LMDs that are physically and functionallydistinct from ‘standard rafts’ and also contain adhesionmolecules (Hemler, 2003). This is of particular interest in viewof recent findings that critically implicate LMDs andtetraspanins (including CD81) in neurite outgrowth and turning(Schmidt et al., 1996; Banerjee et al., 1997; Stipp and Hemler,2000; Guirland et al., 2004). It may suggest SIRP�involvement in growth control.

Fig. 8. Overexpression of cSIRP� affectsaxonal growth in cortical neurons. (A) Theaxons of two neurons cultured on lamininwere visualized by GFP fluorescence (top)and phase-contrast (middle and bottom).(Left) Neuron transfected with GFP only;(right) neuron transfected with cSIRP� plusGFP. The GFP and t=0 phase-contrast imageswere recorded immediately after the additionof IGF-1. Phase-contrast images were alsorecorded 1 hour after the addition of IGF-1(t=1h). The arrows in the images indicate thedistance that the growth cons had advancedduring 1 hour of observation. Note that thegrowth cone shown in the left panelsadvanced much faster than the ones in the right panels. Bar, 10 �m. (B) Quantitation of axon growth rates for neurons cultured on laminin orpoly-D-lysine. Data are shown for neurons transfected with GFP only (control), with cSIRP� plus GFP, or with mutant cSIRP�-FYFF plusGFP. Results are means ± s.e.m. for the number of independent experiments indicated above each bar. Error bars indicate s.e.m. *P<0.05compared to the growth rate measured for control neurons grown on laminin (the left-most bar).

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Involvement of SIRP� in axonal growth controlFor a long time, cell adhesion has been known to affect neuriteoutgrowth (Edgar, 1985; Lander et al., 1985a; Lander et al.,1985b). At least in some cases, this seems to involve Rho A(Liu et al., 2002). In the present report, the role of integrinactivation by laminin is of particular interest. In earlier reports,integrin-dependent phosphorylation of SIRP� has beenassociated with growth control, including that of neurites. Thisseems to involve members of the family of focal adhesionkinases (Tsuda et al., 1998; Oh et al., 1999; Timms et al., 1999;Ivanovic-Dikic et al., 2000). However, it is the Src familykinases that phosphorylate SIRP� in its ITIM motif [whichthen becomes a SHP-2 binding site (Oh et al., 1999)]. Growthcones are exceptionally rich in Src kinases [especially Src andFyn (Maness et al., 1988; Helmke and Pfenninger, 1995)], andgrowth cone Src is known to be activated by integrin bindingto laminin (Helmke et al., 1998). It seemed likely, therefore,that laminin would trigger SIRP� phosphorylation in thegrowth cone, as fibronectin and vitronectin do in fibroblastsand smooth muscle cells (SMCs), respectively (Oh et al., 1999;Maile and Clemmons, 2002b). The growth factors, IGF-1 andBDNF, also are known to increase SIRP� phosphorylation inSMCs (Maile and Clemmons, 2002a) and primary neurons(Ohnishi et al., 1999), respectively. As IGF-1 and BDNF arepotent promoters of axonal growth (in appropriate neurons)(LeRoith et al., 1992; Bibel and Barde, 2000; Pfenninger et al.,2003), a process that is known to be facilitated by laminin(Lander et al., 1985). This may suggest that SIRP� is involvedin the link between growth factor and ECM signals.

We show here that BDNF and, especially, laminin and IGF-1 transiently stimulate SIRP� phosphorylation in growth cones(GCPs contain functional receptors for all three) (Helmke etal., 1998; Pfenninger et al., 2003). Phosphorylation movesSIRP� out of LMDs; new studies will be necessary tounderstand the significance of this dissociation. SIRP�phosphorylation in growth cones requires activity of a Srcfamily kinase and results in temporary increase in theassociation of SHP-2. Phospho-SIRP� is not only a bindingsite for SHP-2, but also a substrate for the phosphatase. Theresulting dephosphorylation of SIRP� (Fujioka et al., 1996;Noguchi et al., 1996) explains the observed biphasic responsesof phosphorylation and SHP-2 association to growth conestimulation with the three factors. Interestingly the kinetics ofSIRP� phosphorylation in GCPs were much faster than thosereported elsewhere (Tsuda et al., 1998; Oh et al., 1999; Ohnishiet al., 1999; Maile and Clemmons, 2002a). Data suggest thatthe initial step of SHP-2 binding to phosphorylated SIRP� alsoserves to activate its phosphatase activity (Ohnishi et al., 1996),which is necessary for the role of SHP-2 in growth factor-activated signal transduction (Kharitonenkov et al., 1997;Araki et al., 2000; Maile and Clemmons, 2002a). In particular,SHP-2 is important for PI 3-kinase activation (Hakak et al.,2000; Wu et al., 2001; Zhang et al., 2002), and PI 3-kinase isnecessary for axonal growth (Laurino et al., 2005). Therefore,transient SIRP� phosphorylation is likely to affect growthfactor signaling in growth cones.

To examine the effects of SIRP� phosphorylation on axonalgrowth, we overexpressed its cytoplasmic domain, cSIRP�, inprimary cortical neurons. Because these neurons require IGF-1 (or high insulin levels sufficient for activating the IGF-1receptor) to maintain outgrowth over extended time periods,

these experiments were done in the presence of IGF-1, whichis known to stimulate membrane addition at the growth cone(Pfenninger et al., 2003). Furthermore, neurons were growneither in the presence or in the absence of integrin activation(laminin versus poly-D-lysine substratum). Soluble cSIRP� isthought to compete with endogenous transmembrane SIRP�for binding to intracellular partners, such as SHP-2. This hasbeen reported to result in a dominant-negative effect inNIH3T3 cells (Neznanov et al., 2003; Neznanov et al., 2004).cSIRP� expressed in cortical neurons was indeedphosphorylated and bound SHP-2. Perhaps surprisingly,cSIRP� expression did not obviously change growth conemorphology, the growth cone cytoskeleton, or the localizationof endogenous SIRP� in primary cortical neurons. However,on laminin cSIRP� reduced the axonal growth rate (in thepresence of IGF-1) by about 67%. Control experimentsinvolving the overexpression of phosphorylation-deficientcSIRP� did not affect the growth rate. This demonstrated theessential role of phosphorylation in the cSIRP� effect onlaminin. We also cultured transfected neurons on poly-D-lysine, which is a more adhesive molecule for these neuronsand resulted in larger growth cones with more numerous andmore pointed, but shorter filopodia. As on laminin, SIRP� wasenriched in these filopodia. For control neurons, the axonalgrowth rate was reduced (to 58%) on poly-D-lysine relative tolaminin. However, upon transfection with cSIRP�, there wasno further reduction of that growth rate. Thus, our datademonstrate that the reduction in growth rate induced byoverexpression of cSIRP� (in the presence of IGF-1) dependson laminin activation of integrin. This suggests that integrin-stimulated SIRP� phosphorylation affects axonal growthprobably by modulating IGF-1 receptor signaling or anothermechanism involving SHP-2 (Neel et al., 2003; Ling et al.,2005).

Although such a response has not been shown in growthcones before, our results are consistent with those obtained inother cell systems, such as NIH3T3 cells (Neznanov et al.,2003) and Smooth muscle cells (Maile and Clemmons, 2002a;Maile and Clemmons, 2002b; Clemmons and Maile, 2005). InSMCs, IGF-1 stimulates SIRP� phosphorylation in thepresence of the �V�3 integrin ligand, vitronectin. This resultsinitially in the binding of SHP-2 to SIRP� and to thesubsequent transfer of SHP-2 to IGF-1 receptor complexes,where signaling is modulated (Maile and Clemmons, 2002a).This alters cellular growth and migration responses to IGF-1.

Overall, growth cone SIRP� appears to be dynamicallyassociated with LMDs probably including growth factorreceptors and integrins (our unpublished observations).Activation of such receptors transiently stimulates, via Srcfamily kinase(s), SIRP� phosphorylation and SHP-2 binding.We show that this affects IGF-1-stimulated axonal growthrates, probably via activation of SHP-2 and modulation of IGF-1 receptor signaling. Thus, SIRP� seems to be necessary forthe modulation of axonal growth by ECM molecules, such aslaminin. As such it may provide at least a partial molecularexplanation for the long-known observation that lamininpromotes growth-factor-stimulated neurite outgrowth (Landeret al., 1985a; Lander et al., 1985b; Edgar et al., 1988; Goldberget al., 2002; Liu et al., 2002). The interaction of SIRP� withCD47 (or other, so far unknown ligands) may further modulategrowth factor responses.

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Materials and MethodsMaterialsTimed pregnant Sprague-Dawley rats were purchased from Harlan Laboratories(Indianapolis, IN). LXSN-GSE2-1 and pcDNA-SIRPc-FYFF L-15 plasmids were akind gift from Nickolay Neznanov, Cleveland Clinic Foundation, OH. Cell culturemedia and supplements, subcloning reagents, pcDNA3.1 vector, mouse laminin,acrylamide and other gel reagents were from Invitrogen (Carlsbad, CA). Tween 20,Triton X-100 (TX100), Brij 98, bovine serum albumin (BSA), methyl-�-cyclodextrin (M�CD), saponin, �-escin, poly-D-lysine, fluorescein isothiocyanate(FITC)-conjugated phalloidin, recombinant mouse insulin-like growth factor (IGF-1), recombinant human brain-derived neurotrophic factor (BDNF), and proteaseinhibitor cocktail were from Sigma (St Louis, MO). Trasylol, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), PP3 (4-amino-7-phenylpyrazol[3,4-d]pyrimidine), protein A/G plus-agarose and genistein were fromCalbiochem (San Diego, CA). Other reagents and their sources were: Alexa Fluor®-conjugated secondary antibodies, Molecular Probes (Eugene, OR); Cy3- and Cy5-conjugated secondary antibodies, Jackson Laboratory (Bar Harbor, ME); anti-SIRP� polyclonal antibody, anti-p85 polyclonal antibody and anti-phospho-tyrosine(pTyr) monoclonal antibody (4G10), Upstate Biotechnology (Lake Placid, NY);anti-SHP2 monoclonal and polyclonal antibodies, Santa Cruz Biotechnology (SantaCruz, CA); anti-Src polyclonal antibody and anti-Src-pY418 monoclonal antibody,Biosource International (Camarillo, CA); pEGFP-N2 and -C2 vectors, anti-CD81monoclonal antibody and anti-pTyr monoclonal antibody (PY20), BD Biosciences(San Jose, CA); fetal bovine serum (FBS), HyClone (Logan, UT); rat neuronNucleofector® kit, Amaxa Biosystems (Köln, Germany); Assistent® cover glasses,Carolina Biological Supply Company (Burlington, NC); SlideBook imagingsoftware, Intelligent Imaging Innovations, Inc. (Denver, CO); BCA protein assayreagent, Pierce (Rockford, IL); anti-SIRP� monoclonal antibody, Serotec (Raleigh,NC); anti-GFP polyclonal antibody (ab290), Abcam (Cambridge, MA);polyvinylidene difluoride (PVDF) membranes, Millipore Corporation (Bedford,MA); ImageQuant 5.2, Molecular Dynamics (Piscataway, NJ); Sykes-Moorechamber, Bellco Glass Inc. (Vineland, NJ); Scion Image 4.0.2, Scion Corp.(Frederick, MD).

Cell culture and transfectionCerebral cortices were dissected from fetal (day 18) Sprague-Dawley rats and cutinto pieces of <1 mm3. These were treated with trypsin (0.5 g/l)-EDTA (2 g/l) for15 minutes at 37°C. Trypsinization was stopped with complete medium (neurobasalcontaining glutamine, glucose, 10% FBS, B27), and tissue was gently triturated (10passages) with glass Pasteur pipettes. Cells were plated in complete medium oneither poly-D-lysine- or laminin-coated cover glasses in 35-mm Petri dishes.Cultures were incubated at 37°C in 5% CO2 in air. Medium was replaced after 6hours with serum-free medium (Neurobasal containing glutamine, glucose, B27)(Brewer et al., 1993). After 48 hours in vitro, long neurites with growth cones wereevident.

To express SIRP� fragment in primary neurons, a pcDNA3.1-cSIRP� vector wasconstructed by inserting an EcoRI fragment from LXSN-GSE2-1 (Neznanov et al.,2003) into pcDNA3.1. Transfection was carried out by electroporation(Nucleofection® system). Briefly, 4.8�106 freshly dissociated cortical cells wereresuspended in 100 �l of Nucleofector solution containing 3 �g plasmid DNA. Forco-transfection experiments, 0.5 �g of plasmid pEGFP-N2 was added with 3 �g ofeither pcDNA3.1-cSIRP� or pcDNA-cSIRP�-FYFF. After electroporation, 500 �lof prewarmed, complete medium was added to the cuvette, and cells weretransferred into culture dishes. Typical transfection efficiencies of surviving neuronswere about 20%.

ImmunofluorescenceCortical cultures were fixed by slow infusion of fixative [4% paraformaldehyde inphosphate-buffered saline (PBS), pH 7.4, 120 mM glucose, 0.4 mM Ca2+] into theculture medium (Pfenninger and Maylie-Pfenninger, 1981). Thereafter, fixative wasremoved by rinsing the cultures with PBS containing 1 mM glycine. Cultures werepermeabilized with 1% Brij 98 in blocking buffer (3% BSA in PBS) for 2 minutesand incubated for 1 hour at room temperature with blocking buffer withoutdetergent. After labeling with primary antibodies (1-3 hours at room temperature)and washing with blocking buffer, cultures were stained with labeled secondaryantibody (Alexa Fluor® 488- or Alexa Fluor® 594-conjugated; 1:200, 1 hour at37°C). We used FITC-conjugated phalloidin (1:200; 1 hour at 37°C) as a label forF-actin. After three washes, the coverslips were mounted onto slides and the culturesobserved by epifluorescence with the Zeiss 63�/1.4 NA oil Plan-Apochromatobjective on a Zeiss Axiovert 200M microscope (phase-contrast images, Zeiss63�/1.25 NA oil Plan-NeoFluar). Digital images were acquired using a CookeSensicam camera and processed with SlideBook software. Optical sections weretaken at 0.1-0.2 �m intervals and deconvolved using a nearest-neighbor algorithm.

Growth cone isolationBrains from E18 fetal rats were homogenized in 0.32 M sucrose buffer (0.32 Msucrose, 100 KIU/ml Trasylol (aprotinin), 1 mM MgCl2 and 1 mM TES, pH 7.3)and centrifuged at 3000 g for 15 minutes to generate a low-speed supernatant (LSS).

LSS was loaded on a discontinuous density gradient consisting of 0.83, 1.2 and 2.66M sucrose. The 0.32 M/0.83 M interface contained the isolated growth cones orgrowth cone particles (GCPs) (Pfenninger et al., 1983; Lohse et al., 1996).

Gel electrophoresis and western blottingProtein concentrations were determined using BCA protein assay reagent withBSA as the standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was performed essentially as described previously (Laemmli, 1970).Gels were run under reducing conditions except those for CD81. Separatedpolypeptides were electrotransferred onto PVDF membranes in Tris-glycine buffercontaining 15% methanol. Blots were quenched with 5% fat-free milk powder inTris-buffered saline (TBS; 10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing0.2% Tween 20 (TBST), for 1 hour at room temperature. Incubation with primaryantibody, in TBST/5% milk powder, was for 2 hours at room temperature. Afterwashing, the blots were incubated with Cy3- or Cy5-conjugated secondaryantibody in TBST for 1 hour at room temperature. After washing, bound antibodywas imaged with a Typhoon® 9400 laser scanner (General Electric). The imageswere analyzed by ImageQuant 5.2 software. Sometimes, data were collected inboth the Cy5 and the Cy3 channels for simultaneous quantification of two labeledpolypeptides.

Lipid microdomain analysisGCPs were extracted for 5 minutes with 1% TX100 (w/v) or 1% Brij 98 (w/v), onice or at 37°C, respectively. Solubilized samples were mixed with 2 M sucrose (finalconcentration, 1.33 M) and chilled on ice before being placed at the bottom of astep gradient (1.0 M, 0.9 M, 0.7 M, 0.5 M sucrose). Gradients were centrifuged at200,000 g for 16 hours at 4°C. Fractions (0.5-ml) were collected and proteinprecipitated with methanol/chloroform (Wessel and Flugge, 1984). Pellets wereanalyzed by SDS-PAGE and western blotting.

For cholesterol sequestration GCPs were pre-treated with 0.2% saponin (w/v) at4°C, or with 10 mM M�CD at 37°C, for 30 minutes before detergent extraction.

Neurite growth rate measurementsAfter 48 hours, in vitro-transfected neurons were identified by their greenfluorescence. Neurite growth rates were assessed by time-lapse imaging. Coverglasses mounted into Sykes-Moore chambers were covered with serum-free,modified B27 neurobasal medium without insulin. 10 nM IGF-1 was added andthe medium overlaid with mineral oil (embryo-tested; Sigma) to maintain pH andprevent evaporation. The chamber was kept on the pre-heated microscope stageunder convective heating at 37°C. Neurons were imaged at 15-minute intervalsfor 2-3 hours (Zeiss Axiovert 200 M microscope, 20�/0.5 NA or 40�/0.75 NAPlan-NeoFluar objectives; Cooke Sensicam camera). With Scion Image 4.0.2 wemeasured the distance between the center of growth cones and the center of theneuronal perikarya or the point where the neurite entered the field. Neurites(presumably, axons) had to meet the following criteria (Lemmon et al., 1989):longest process of the neuron before and after the measurement; tipped by asingle growth cone; growth unobstructed and without branching; no majorretraction.

Phosphorylation assay in isolated growth conesThe GCP suspension from the gradient was mixed with an equal volume of‘intracellular buffer’ (20 mM Hepes pH 7.3, 50 mM KCl, 5 mM NaCl, 3 mMMgCl2), permeabilized with 0.01% �-escin, and incubated for 5 minutes on ice, inthe presence or absence of factor or other reagent (laminin, 35 �g/ml; BDNF, 0.2nM; IGF-1, 1 nM; PP2, 1 �M; PP3, 1 �M). Upon the addition of 1 mM ATP, GCPswere warmed to 37°C for 1 or 5 minutes. Usually, the reaction was terminated bychilling and adding 1% TX100 plus 3 mM vanadate, 2 mM NaF, 10 mM EDTA,100 �M genistein and protease-inhibitor cocktail. After 10 minutes on ice, Triton-insoluble elements were pelleted at 30,000 g for 1 hour. SIRP� wasimmunoprecipitated from this supernatant with anti-SIRP� (6 �g/ml) for 2 hoursat 4°C before adding protein A- and G-coated beads. Fresh vanadate was added toevery solution change. Precipitates were resolved by SDS-PAGE, blotted, andprobed with anti-pTyr to reveal SIRP� phosphorylation, and with anti-SHP2 toexamine SHP2 association. In LMD experiments, reactions were stopped as above,but without adding TX100, and samples were extracted with 1% Brij 98. In somecases the reaction was terminated by methanol/chloroform precipitation. To detectactivated Src, the resulting pellets were analyzed by western blot using phospho-Src-specific antibody (anti-Src-pY418).

Immunoprecipitation of cSIRP� from culturecSIRP� was inserted into the pEGFP-C2 vector for expression of GFP-cSIRP�fusion protein in cultured cortical neurons. Forty-eight hours post-transfection,cultures were treated with medium with or without 1 mM vanadate for 10 minutesand cells were scraped off and homogenized. A low-speed supernatant of harvestedmaterial was permeabilized with 0.01% �-escin and incubated with or without 1mM ATP plus vanadate at 37°C for 10 minutes, followed by the addition of 1%TX100 and protease inhibitors. After 10 minutes on ice, samples were centrifugedat 10,000 g for 10 minutes and GFP-cSIRP� was immunoprecipitated from thesupernatant with anti-GFP antibody as described above.

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The authors wish to thank Nicolay Neznanov of Cleveland ClinicFoundation, Cleveland, OH, USA for the generous gift of wild-typeand mutant SIRP� tail constructs. This work was supported by NIHgrant R01 NS41029.

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