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PubMed2001

The voltage-gated Ca2+ channel is the Ca2+ sensor of fast neurotransmitter release.

Previously it demonstrated that in the absence of Ca2+ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca2+]i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217-220.). These studies designate Ca2+ entry as opposed to [Ca2+]i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca+ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein-protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J 15:4100-4110; Wiser, O., Trus, M.. Hernandez, A., Renström, E., Barg, S., Rorsman. P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248-253). The kinetics of Ca(v)1.2 (Lc-type) and Ca(v)2.2 (N-type) Ca2+ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin 1, a vesicle-associated protein, accelerated the activation kinetics indicating Ca(v)2.2 coupling to the vesicle. The unique modifications of Ca(v)1.2 and Ca(v)2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca(v)1.2 cytosolic domain Lc(753-893), acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca2+ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca2+-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca2+-dependent CAV (RRP) release, initiated by the binding of Ca2+ to the channel, upstream to intracellular Ca2+ sensor thus establishing the Ca2+ channel as the Ca2+ sensor of neurotransmitter release.

Animals↗

PubMed2000

Expression of presynaptic proteins is closely correlated with the chronotopic pattern of axons in the retinotectal system of the chick.

Newly synthesized presynaptic integral membrane proteins in neurons are transported in precursor vesicles from the site of protein biosynthesis in the cell body by fast axonal flow to the presynaptic terminal. We followed the path that presynaptic proteins travel on the way to their central targets of the highly ordered primary visual pathway of the chick and analyzed the developmental changes in the expression of synaptic vesicle protein 2 (SV2), synaptotagmin, and syntaxin. Immunofluorescences revealed that: (1) the onset of protein expression in the retinal ganglion cells occurs in a central to peripheral developmental pattern from embryonic day 4 (E4) onward; (2) the proteins were found first in the inner and later in the outer plexiform layer of the retina; and (3) they were redistributed from the photoreceptor inner segments and cell bodies to the terminals in the outer plexiform layer. From E4 onward, immunopositive axons for SV2, synaptotagmin, and syntaxin were found in the optic nerve, disappearing after E9 for SV2 and synaptotagmin. The optic tract was stained for SV2 and synaptotagmin between E7 and E12, for syntaxin until the posthatching period. Finally, immunoreactivities for the investigated proteins were present at the surface of the tectum from E8 onward, when first retinal axons arrived there. The present study revealed that SV2 and synaptotagmin, but not syntaxin, are, expressed in a transient wave that follows the advancement of optic axons and the proteins towards the optic tectum.

Animals↗

PubMed1998

Differential expression of rat brain synaptic proteins in development and aging.

We have previously reported the differential involvement of synaptic proteins in Alzheimer's disease (AD). As AD is an aging-associated disease, in the present study we examined the developmental and aging-related changes in synaptic proteins such as synaptophysin, synaptobrevin, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), syntaxin 1/HPC-1 and drebrin in the rat brain. Immunoblot analyses of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that the protein level of synaptophysin and synaptobrevin increased after birth, being highest at 24 weeks, and then decreased with aging. Synaptotagmin was detected at E19, with levels increasing after birth to 96 weeks. SNAP-25 levels were highest at 4 weeks, and then decreased with aging. Syntaxin 1/HPC-1 levels were high at E19 and 1 week, decreasing rapidly from 2 weeks onwards, and drebrin levels were highest at E19 and 1 week, and decreased during aging. The present results suggest that the expression of each synaptic protein is differentially regulated in development and aging.

Aging↗

PubMed2002

Modulation of a brain voltage-gated K+ channel by syntaxin 1A requires the physical interaction of Gbetagamma with the channel.

Recently we suggested that direct interactions between voltage-gated K(+) channels and proteins of the exocytotic machinery, such as those observed between the Kv1.1/Kvbeta channel, syntaxin 1A, and SNAP-25 may be involved in neurotransmitter release. Furthermore, we demonstrated that the direct interaction with syntaxin 1A enhances the fast inactivation of Kv1.1/Kvbeta1.1 in oocytes. Here we show that G-protein betagamma subunits play a crucial role in the enhancement of inactivation by syntaxin 1A. The effect caused by overexpression of syntaxin 1A is eliminated in the presence of chelators of endogenous betagamma subunits in the whole cell and at the plasma membrane. Conversely, enhancement of inactivation caused by overexpression of beta(1)gamma(2) subunits is eliminated upon knock-down of endogenous syntaxin or its scavenging at the plasma membrane. We further show that the N terminus of Kv1.1 binds brain synaptosomal and recombinant syntaxin 1A and concomitantly binds beta(1)gamma(2); the binding of beta(1)gamma(2) enhances that of syntaxin 1A. Taken together, we suggest a mechanism whereby syntaxin and G protein betagamma subunits interact concomitantly with a Kv channel to regulate its inactivation.

Animals↗

PubMed2002

Effect of hypothyroidism on synaptosomal-associated protein of 25 kDa and syntaxin-1 expression in adenohypophyses of rat.

The aim of this study was to analyze the expression of SNAP-25 and syntaxin-1 in the adenohypophyses of hypothyroid rats. Rats were divided into: 1) controls; 2) thyroidectomized 40 days; and 3) thyroidectomized 40 days with replacement of T4 20 days after surgery. Adenohypophyses were studied by immunohistochemistry and immunoblot analysis using antibodies against SNAP-25, syntaxin-1 and TSH. By immunostaining, SNAP-25 and syntaxin-1 were conspicuous and were localized around cytoplasmic vacuoles of thyroidectomy cells. Immunoblot analysis shows that thyroidectomy increases adenohypophysial SNAP-25 expression and decreases syntaxin-1 levels. T4 administration for 20 days produces a recovery similar to control values. In conclusion, thyroidectomy produces changes in both expression and immunoreactivity of SNAP-25 and syntaxin-1 in adenohypophyses of rats and these effects can be reversed by T4 administration.

Animals↗

PubMed2000

Participation of syntaxin 1A in membrane trafficking involving neurite elongation and membrane expansion.

Syntaxin 1A has been implicated to play an important role in neurotransmitter release by regulating synaptic vesicle fusion. The protein is also suggested to be required for other types of membrane fusion such as cellularization during embryonic development. In the current study, we overexpressed syntaxin 1A, SNAP-25b, and VAMP-2 in PC12 cells using recombinant adenoviruses, and determined their effects on membrane trafficking involving neurite outgrowth. It was found that overexpression of syntaxin 1A inhibited NGF-induced neurite extension, and the expressed syntaxin was localized to the plasma membrane, intracellular membranes, and the neurite tips. SNAP-25 overexpression slightly enhanced neurite elongation, whereas no significant changes in neurite growth was observed in VAMP-overexpressing cells. The effect of syntaxin 1A in general membrane trafficking was further studied by transient transfection of non-neuronal cells. Syntaxin 1A expression in HEK 239 and NIH3T3-L1 caused the cells to lose their normal morphology, leading to round and smaller cells. Deletion of the C-terminal sequence containing the H3 helix and the membrane anchoring domains of syntaxin abolished its ability to induce cell morphology changes, whereas removal of the N-terminal 1-170 amino acid sequence did not affect this activity. These findings suggest that in addition to its well documented role in synaptic vesicle fusion, syntaxin may function in other non-synaptic membrane trafficking such as neurite outgrowth and membrane expansion.

3T3 Cells↗

PubMed2004

Syntaxin-1A binds to and modulates the Slo calcium-activated potassium channel via an interaction that excludes syntaxin binding to calcium channels.

From its position in presynaptic nerve terminals, the large conductance Ca(2+)-activated K+ channel, Slo, regulates neurotransmitter release. Several other ion channels known to control neurotransmitter release have been implicated in physical interactions with the neurotransmitter release machinery. For example, the Ca(v)2.2 (N-type) Ca2+ channel binds to and is modulated by syntaxin-1A and SNAP-25. Furthermore, a close juxtaposition of Slo and Ca(v)2.2 is presumed to be necessary for functional coupling between the two channels, which has been shown in neurons. We report that Slo exhibits a strong association with syntaxin-1A. Robust co-immunoprecipitation of Slo and syntaxin-1A occurs from transfected HEK293 cells as well as from brain. However, despite this strong interaction and the known association between syntaxin-1A and the II-III loop of Ca(v)2.2, these three proteins do not co-immunoprecipitate in a trimeric complex from transfected HEK293 cells. The Slo-syntaxin-1A co-immunoprecipitation is not significantly influenced by [Ca2+]. Multiple relatively weak interactions may sum up to a tight physical coupling of full-length Slo with syntaxin-1A: the C-terminal tail and the S0-S1 loop of Slo each co-immunoprecipitate with syntaxin-1A. The presence of syntaxin-1A leads to reduced Slo channel activity due to an increased V(1/2) for activation in 100 nM, 1 muM, and 10 microM Ca2+, reduced voltage-sensitivity in 1 microM Ca2+, and slower rates of activation in 10 microM Ca2+. Potential physiological consequences of the interaction between Slo and syntaxin-1A include enhanced excitability through modulation of Slo channel activity and reduced neurotransmitter release due to disruption of syntaxin-1A binding to the Ca(v)2.2 II-III loop.

Antigens, Surface↗

PubMed2000

Identification of syntaxin 1A as a novel binding protein for presenilin-1.

Mutations in the presenilin 1 gene have been shown to result in Alzheimer's disease. Presenilin 1 is a multi-transmembrane protein with a large hydrophilic loop near the C-terminus. This region is required for known functions of presenilin 1. We have constrained this loop within the active site of the bacterial protein, thioredoxin, to mimic its native conformational state. This hybrid protein was used as bait in a yeast two hybrid screen in an attempt to identify presenilin binding proteins. By this method syntaxin 1A, a synaptic plasma membrane protein, was identified as a novel binding protein for presenilin 1. In vitro experiments confirm the two-hybrid results suggesting that PS1 binds syntaxin under physiological conditions.

Alzheimer Disease↗

PubMed2003

Syntaxin 1A co-associates with native rat brain and cloned large conductance, calcium-activated potassium channels in situ.

Large conductance, calcium-activated potassium channels (BKCa channels) are regulated by several distinct mechanisms, including phosphorylation/dephosphorylation events and protein-protein interactions. In this study, we have examined the interaction between BKCa channels and syntaxin 1A, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is reported to modulate the activity and/or localization of different classes of ion channels. Using a reciprocal co-immunoprecipitation strategy, we observed that native BKCa channels in rat hippocampus co-associate with syntaxin 1A, but not the closely related homologue syntaxin 3. This BKCa channel-syntaxin 1A interaction could be further demonstrated in a non-neuronal cell line (human embryonic kidney (HEK) 293 cells) following co-expression of rat syntaxin 1A and BKCa channels cloned from either mouse brain or bovine aorta. However, co-expression of these same channels with syntaxin 3 did not lead to a detectable protein-protein interaction. Immunofluorescent co-staining of HEK 293 cells expressing BKCa channels and syntaxin 1A demonstrated overlapping distribution of these two proteins in situ. Functionally, co-expression of BKCa channels with syntaxin 1A, but not syntaxin 3, was observed to enhance channel gating and kinetics at low concentrations (1-4 microM) of free cytosolic calcium, but not at higher concentrations (< or = 10 microM), as judged by macroscopic current recordings in excised membrane patches. Interactions between BKCa channels and neighbouring membrane proteins may thus play important roles in regulating the activity and/or distribution of these channels within specialized cellular compartments.

Animals↗

PubMed2006

Syntaxin 1A promotes the endocytic sorting of EAAC1 leading to inhibition of glutamate transport.

The neuronal glutamate transporter, excitatory amino-acid carrier 1 (EAAC1), plays an important role in the modulation of neurotransmission and contributes to synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and to epileptogenesis. However, the mechanisms that regulate EAAC1 endocytic sorting and function remain largely unknown. Here, we first demonstrate that EAAC1 undergoes internalization through the clathrin-mediated pathway and further show that syntaxin 1A, a key molecule in synaptic exocytosis, potentiates EAAC1 internalization, thus leading to the functional inhibition of EAAC1. In the presence of the transmembrane domain of syntaxin 1A, its H3 coiled-coil domain of syntaxin 1A is necessary and sufficient for the inhibition of EAAC1. Furthermore, specific suppression of endogenous syntaxin 1A significantly blocked EAAC1 endocytic sorting and lysosomal degradation promoted by kainic acid, a drug for kindling the animal model of human temporal lobe epilepsy in rat, indicating a potential role of syntaxin 1A in epileptogenesis. These findings provide new evidence that syntaxin 1A serves as an intrinsic enhancer to EAAC1 endocytic sorting and further suggest that syntaxin 1A is conversant with both ;ins' and ;outs' of synaptic neurotransmission.

Amino Acid Transport Systems↗

PubMed1997

Developmental expression of synaptophysin, synapsin I and syntaxin in the rat retina.

Expression of synaptophysin, synapsin I and syntaxin was studied immunocytochemically in the developing rat retina using indirect immunoperoxidase technique. In the inner plexiform layer (IPL), syntaxin immunoreactivity appeared at postnatal day 1 (P1) whereas synaptophysin and synapsin I staining were first observed at P2. In the outer plexiform layer (OPL), synaptophysin appeared at P4, while synapsin I and syntaxin appeared at P8. In the case of synaptophysin, a punctate pattern of staining was observed from the time of its appearance (P4) in the OPL and from P12 onwards in the IPL. Synapsin I and syntaxin immunoreactivity in the OPL were of a low intensity throughout the development and in the adult stage. These findings are discussed in relation to synaptogenesis in the rat retina.

Animals↗

PubMed2004

The plasma membrane Q-SNARE syntaxin 2 enters the zymogen granule membrane during exocytosis in the pancreatic acinar cell.

During exocytosis in the pancreatic acinar cell, zymogen granules fuse directly with the apical plasma membrane and also with granules that have themselves fused with the plasma membrane. Together, these primary and secondary fusion events constitute the process of compound exocytosis. It has been suggested that the sequential nature of primary and secondary fusion is a consequence of the requirement for plasma membrane soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors, such as syntaxin 2, to enter the membrane of the primary fused granule. We have tested this possibility by determining the location of syntaxin 2 in unstimulated and stimulated pancreatic acini. Syntaxin 2 was imaged by confocal immunofluorescence microscopy. Fused granules were detected both through their filling with the aqueous dye lysine-fixable Texas Red-dextran and through the decoration of their cytoplasmic surfaces with filamentous actin. In unstimulated cells, syntaxin 2 was exclusively present on the apical plasma membrane. In contrast, after stimulation, syntaxin 2 had moved into the membranes of fused granules, as judged by its location around dye-filled structures of 1-mum diameter that were coated with filamentous actin. At long times of stimulation (5 min), the majority (85%) of dye-filled granules were also positive for syntaxin 2. In contrast, at shorter times (1 min), more dye-filled granules (29%) were syntaxin 2-negative. We conclude that syntaxin 2 enters the membrane of a fused zymogen granule after the opening of the fusion pore, and we suggest that this movement might permit the onset of secondary fusion.

Actins↗

PubMed2004

Syntaxin 5 interacts with presenilin holoproteins, but not with their N- or C-terminal fragments, and affects beta-amyloid peptide production.

Mutations in presenilins 1 and 2 (PS1 and PS2) account for the majority of cases of early-onset familial Alzheimer's disease. However, the trafficking and interaction of PSs with other proteins in the early secretory pathways are poorly understood. Using co-immunoprecipitation, we found that PS bound to Syx5 (syntaxin 5), which is a target-soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor involved in endoplasmic reticulum (ER)-Golgi vesicular transport in vivo. Syx5 interacted only with the full-length PS holoproteins and not with the naturally occurring N- or C-terminal fragments. The PS holoproteins co-immunoprecipitated with the mutant Syx5, which localized to the ER and Golgi compartments, despite the substitution of the transmembrane region with that of syntaxin 1A. In contrast, the transmembrane deletion mutant that localized to the cytosol, but not to the ER or Golgi compartments, did not co-immunoprecipitate the PS holoproteins. The PS1 variant linked to familial Alzheimer's disease (PS1DeltaE9), lacking the region that contains the endoproteolytic cleavage site in the cytoplasmic loop, showed markedly decreased binding to Syx5. Immunofluorescence and sucrose-density-gradient fractionation analyses showed that the full-length PS holoproteins co-localized with Syx5 to the ER and cis-Golgi compartments. Furthermore, Syx5 overexpression resulted in the accumulation of PS holoproteins and the beta-amyloid precursor protein, and reduced the secretion of the Abeta (amyloid beta) peptide in COS-7 cells. In summary, these results indicate that Syx5 binds to full-length PSs and affects the processing and trafficking of beta-amyloid precursor protein in the early secretory compartments.

Amyloid beta-Peptides↗

PubMed2002

Perturbation of a very late step of regulated exocytosis by a secretory carrier membrane protein (SCAMP2)-derived peptide.

Secretory carrier membrane proteins (SCAMPs) are conserved four transmembrane-spanning proteins associated with recycling vesicular carriers. In mast cells, as in other cell types, SCAMPs 1 and 2 are present in secretory granule membranes and other intracellular membranes. We now demonstrate a population of these SCAMPs in plasma membranes. Although small, this population partially colocalizes with SNARE proteins SNAP-23 and syntaxin 4. A fraction of SCAMPs 1 and 2 also coimmunoprecipitates with SNAP-23. An oligopeptide, E peptide, within the cytoplasmic segment linking the second and third transmembrane spans, particularly of SCAMP2, potently inhibits exocytosis in streptolysin O-permeabilized mast cells. The E peptide is unique to SCAMPs and highly conserved among SCAMP isoforms, and minor changes in its sequence abrogate inhibition. It blocks fusion beyond the putative docking step where granules contact the cell surface and each other during compound exocytosis. Blockade is also beyond Ca(2+)/ATP-dependent relocation of SNAP-23, which regulates compound exocytosis, and beyond ATP-dependent priming of fusion. Kinetic ordering of exocytotic inhibitors has shown that E peptide acts later than other perturbants at a stage closely associated with membrane fusion. These findings identify a new reagent for analyzing the final stage of exocytosis and point to the likely action of SCAMP2 in this process.

Amino Acid Sequence↗

PubMed2005

Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion.

At low surface concentrations that permit formation of impermeable membranes, neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins form a stable, parallel, trans complex when vesicles are brought into contact by a low concentration of poly(ethylene glycol) (PEG). Surprisingly, formation of a stable SNARE complex does not trigger fusion under these conditions. However, neuronal SNAREs do promote fusion at low protein/lipid ratios when triggered by higher concentrations of PEG. Promotion of PEG-triggered fusion required phosphatidylserine and depended only on the surface concentration of SNAREs and not on the formation of a trans SNARE complex. These results were obtained at protein surface concentrations reported for synaptobrevin in synaptic vesicles and with an optimally fusogenic lipid composition. At a much higher protein/lipid ratio, vesicles joined by SNARE complex slowly mixed lipids at 37 degrees C in the absence of PEG, in agreement with earlier reports. However, vesicles containing syntaxin at a high protein/lipid ratio (>or=1:250) lost membrane integrity. We conclude that the neuronal SNARE complex promotes fusion by joining membranes and that the individual proteins syntaxin and synaptobrevin disrupt membranes so as to favor formation of a stalk complex and to promote conversion of the stalk to a fusion pore. These effects are similar to the effects of viral fusion peptides and transmembrane domains, but they are not sufficient by themselves to produce fusion in our in vitro system at surface concentrations documented to occur in synaptic vesicles. Thus, it is likely that proteins or factors other than the SNARE complex must trigger fusion in vivo.

Liposomes↗

PubMed1998

Functional analysis of the C2A domain of synaptotagmin 1: implications for calcium-regulated secretion.

Synaptotagmin 1 is proposed to function as a low affinity calcium sensor for calcium-triggered exocytosis from neural and neuroendocrine cells. Because of the calcium-binding properties of the C2A domain of synaptotagmin 1, calcium-dependent interactions through this domain may modulate neurotransmitter release. We addressed this question by using alanine-scanning mutagenesis to generate a series of mutations within the C2A domain of synaptotagmin 1. The effects of these mutations on synaptotagmin 1 C2A function were analyzed for (1) calcium-dependent phospholipid binding, (2) calcium-dependent binding to syntaxin 1A, a plasma membrane protein critical for vesicle docking or fusion, and (3) calcium-regulated secretion after microinjection into neuroendocrine pheochromocytoma (PC12) cells. Our analyses reveal that a polylysine motif at residues 189-192 confers an inhibitory effect on secretion by recombinant synaptotagmin C2A fragments. The synaptotagmin 1 C2A polylysine motif functions independently of calcium-mediated interactions with phospholipids and syntaxin 1A. Furthermore, alpha-latrotoxin reverses the inhibitory effect of injected recombinant C2A fragments, suggesting that they perturb the cellular calcium-sensing machinery by interfering with synaptotagmin 1 activity in vivo. Our results indicate that novel calcium-independent interactions mediated through the C2A polylysine motif of synaptotagmin 1 function to modulate neurotransmitter release.

Animals↗

PubMed2001

Syntaxin 1A supports voltage-dependent inhibition of alpha1B Ca2+ channels by Gbetagamma in chick sensory neurons.

N-type Ca(2+) channels are modulated by a variety of G-protein-coupled pathways. Some pathways produce a transient, voltage-dependent (VD) inhibition of N channel function and involve direct binding of G-protein subunits; others require the activation of intermediate enzymes and produce a longer-lasting, voltage-independent (VI) form of inhibition. The ratio of VD:VI inhibition differs significantly among cell types, suggesting that the two forms of inhibition play unique physiological roles in the nervous system. In this study, we explored mechanisms capable of altering the balance of VD and VI inhibition in chick dorsal root ganglion neurons. We report that (1) VD:VI inhibition is critically dependent on the Gbetagamma concentration, with VI inhibition dominant at low Gbetagamma concentrations, and (2) syntaxin-1A (but not syntaxin-1B) shifts the ratio in favor of VD inhibition by potentiating the VD effects of Gbetagamma. Variations in expression levels of G-proteins and/or syntaxin provide the means to alter over a wide range both the extent and the rate of Ca(2+) influx through N channels.

Alternative Splicing↗

PubMed1997

Expression of p67 (Munc-18), Cdk5, P-NFH and syntaxin during development of the rat cerebellum.

Cyclin-dependent kinases (cdks), which regulate the cell division cycle, have also been found in postmitotic neurons. Cdk5, isolated from neural tissue, has been shown to phosphorylate neurofilaments (NFs). Instead of cyclins, however, other neuron-specific activators of cdk5 have been identified including a 67-kD protein (p67) which is identical to a syntaxin-binding protein (n-sec-1, Munc 18) that is thought to play a role in synaptic vesicle trafficking and transmitter release. These functions for p67 are not mutually exclusive since regulation of edk5 phosphorylation of cytoskeletal proteins may modulate axonal dynamics during growth, synaptogenesis and vesicle transport. To gain further insight into the role of p67 in neural tissue, we carried out a Western blot and immunohistochemical analysis of the developing rat cerebellum using antibodies to cdk5, p67, syntaxin and phosphorylated and nonphosphorylated neurofilaments. We assumed that spatiotemporal colocalizations of antigens might correlate with proposed functions for p67. The immunoblots showed that all antigens were developmentally regulated, and increased in expression from PN2 to the adult, with p67 and cdk5 showing a close temporal correlation. Immunohistochemically, p67 colocalized with cdk5 and P-NFH in selected fiber tracts, particularly those in the deep cerebellum. For the most part, p67 also showed strong colocalization patterns with syntaxin in regions of synaptogenesis throughout development such as the molecular layer and glomeruli of the inner nuclear layer. Finally, certain fiber tracts, the afferent fibers, climbing and mossy fibers and particularly the basket cell fibers that envelop and innervate Purkinje cell somata and dendrites, displayed colocalization of cdk5 and P-NFH without expressing any p67. Given the limitations of colocalization data in defining functional relationships, the results are consistent with the hypothesis that p67 is a multifunctional protein, its activity during cerebellum development dependent upon the neuronal phenotype, its location and its state of developmental maturation.

Animals↗