ARTICLE |
Correspondence to: Peter Redecker, Abt. Anatomie 1, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. E-mail: redecker.peter@mh-hannover.de
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proline-rich synapse-associated protein-1 (ProSAP1) is a neuronal PDZ domain-containing protein that has recently been identified as an essential element of the postsynaptic density. Via its interaction with the actin-binding protein cortactin and its integrative function in the organization of neurotransmitter receptors, ProSAP1 is believed to be involved in the linkage of the postsynaptic signaling machinery to the actin-based cytoskeleton, and may play a role in the cytoskeletal rearrangements that underlie synaptic plasticity. As a result of our ongoing studies on the distribution and function of this novel PDZ domain protein, we now report that the expression of ProSAP1 is restricted neither to neurons and interneuronal junctions nor to the nervous system. Using immunohistochemical techniques in conjunction with specific antibodies, we found that, in the CNS, ProSAP1 can be detected in certain glial cells, such as ependymal cells, tanycytes, subpial/radial astrocytes, and in the choroid plexus epithelium. Moreover, our immunohistochemical analyses revealed the presence of ProSAP1 in endocrine cells of the adenohypophysis and of the pancreas, as well as in non-neuronal cell types of other organs. In the pancreas, ProSAP1 immunoreactivity was also localized in the duct system of the exocrine parenchyma. Our findings demonstrate that, in addition to neurons, ProSAP1 is present in various non-neuronal cells, in which it may play a crucial role in the dynamics of the actin-based cytoskeleton. (J Histochem Cytochem 49:639648, 2001)
Key Words: PSD, shank, CortBP1, actin cytoskeleton, glial cells, choroid plexus, pituitary, endocrine pancreas, pancreatic ducts, apical surfaces
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During recent years, many studies have aimed at deciphering the molecular architecture of the postsynaptic density (PSD). The PSD is an electron-dense structural matrix beneath the postsynaptic membrane, which is believed to organize the postsynaptic signal transduction machinery and which probably plays a crucial role for synaptic plasticity (
The recently described PSD protein ProSAP1 (proline-rich synapse-associated protein-1), also known as cortactin-binding protein-1 (CortBP1;
Although the molecular dissection of the PSD is naturally focused on neurons, a full understanding of the PSD at the molecular level also requires a detailed knowledge of the possible extraneuronal distribution and function of its protein constituents, even if the PSD is usually regarded as a unique trait of neurons. In this context, it should be recalled that it is already well known that many pre- and postsynaptic proteins of the synaptic signal transduction apparatus are not restricted to neurons. For example, in the CNS several proteins implicated in the regulation of synaptic vesicle trafficking could readily be detected in glial cells (
In the present immunohistochemical and immunochemical study, we therefore sought to further characterize the novel postsynaptic protein ProSAP1 by asking (a) whether the expression of ProSAP1 in the CNS is really confined to neurons, and (b) whether this protein is also detectable in non-neuronal cells, especially in endocrine cells such as those of the adenohypophysis and of pancreatic islets. The latter endocrine organs were chosen for a detailed investigation because they are already known to be endowed with various protein components involved in presynaptic function.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and Tissue Preparation for Immunohistochemistry
Adult Wistar and Lewis rats of both sexes, which had been kept under normal laboratory conditions, were used for the investigation. Principles of laboratory animal care and specific national laws were followed. Anesthetized animals were transcardially perfused with prewash and fixative solutions, as detailed elsewhere (
Immunohistochemistry
Antibodies.
We used three different antibodies against ProSAP1 which have been characterized in detail in a previous study (
Immunohistochemical Protocol.
Sections were processed for the avidinbiotinperoxidase complex (ABC) technique or for immunofluorescence staining as outlined previously (
Method controls generally consisted of omission of single steps in the immunohistochemical protocol, application of primary antibodies of unrelated specificities or of non-immune sera, use of ascending dilutions of the first antibody, or use of high-molar (0.5 M) PBS as a rinsing solution between the various steps of the immunohistochemical protocol.
Immunoblotting.
Electrophoresis and immunoblotting of total tissue lysates or of crude synaptosomal fractions of the tissues under investigation were performed essentially as reported previously (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry
Our immunohistochemical study revealed a punctate pattern of immunoreactivity in many regions of the rat brain, compatible with the predominant synaptic localization of ProSAP1 described previously (
|
|
Our immunohistochemical investigation also disclosed the presence of ProSAP1 in endocrine cells. In the adenohypophysis, many ProSAP1-immunoreactive cells were distributed throughout the anterior lobe (Fig 3A), whereas only faint immunoreactivity could be observed in the intermediate lobe. In addition, strong immunoreactivity was detectable in the non-endocrine marginal layer cells lining the hypophyseal cleft (not shown). In most ProSAP1-positive adenohypophyseal cells, immunostaining was visible in the whole cytoplasm, including the cell cortex, and frequently appeared in a punctate pattern (Fig 3A, Fig 3C, and Fig 3D). For identification of the immunopositive anterior lobe cells, serial semithin sections were alternatively immunostained for pituitary hormones or ProSAP1. We found that somatotrophs were most densely stained by the ProSAP1 antibodies (Fig 3D and Fig 3E), whereas gonadotrophs expressing LH and/or FSH displayed only a moderate degree of immunoreactivity. Thyrotrophs, lactotrophs, and corticotrophs were at most faintly immunopositive or appeared unreactive to the ProSAP1 antibodies.
|
ProSAP1-positive endocrine cells were also discernible in the pancreas, where all islets in the sections were clearly immunostained. Analysis of serial semithin sections demonstrated that ProSAP1 was expressed in both insulin-producing B-cells (Fig 4A and Fig 4B) and glucagon-producing A-cells (not shown), whereas somatostatin-positive D- and pancreatic polypeptide-positive PP-cells were only weakly immunoreactive. In contrast to ProSAP1, in both adenohypophyseal and islet cells cortactin (Fig 3B and Fig 4G) and actin were concentrated in the subplasmalemmal cytoplasm. Interestingly, in the pancreas expression of ProSAP1 was not restricted to the endocrine compartment. In the exocrine parenchyma, ProSAP1 was consistently localized in the duct system. Pronounced immunostaining for ProSAP1 was conspicuous in the luminal cell cortex of centroacinar, intercalated duct, and intra/interlobular duct cells (Fig 4C4E). Similarly, dense immunostaining in the apical cytoplasm of these epithelial cells could be elicited by antibodies against cortactin (Fig 4F) and actin. Double labeling revealed the co-distribution of the latter proteins and ProSAP1 in the luminal cell cortex (not shown).
|
In addition to the organs on which we focused in this study, we also noted immunoreactivity for ProSAP1 in other tissues, such as the epithelium of hepatic bile ducts, of the tubule system in the kidney, and the bronchial/bronchiolar tree of the lung.
Immunoblotting
Similar to previously published results (180 kD to
230 kD on immunoblots of brain tissue (Fig 5). Protein bands in the same molecular weight range were also revealed on immunoblots of pituitary and pancreatic homogenates (Fig 5A) and of other non-neuronal tissues such as lung, liver, testis, and heart (Fig 5B). Furthermore, corresponding bands became visible in blots of kidney and skeletal muscle homogenates after longer film exposure.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ProSAP1 and the closely related molecule ProSAP2 are multidomain proteins that are believed to be central elements of the PSD protein network. Established interactions of both proteins comprise the association with the actin-binding protein cortactin and the binding to members of the GKAP/SAPAP protein family (
Our immunohistochemical analysis has revealed that ProSAP1 is present in ependymal cells and tanycytes of the cerebral ventricles and of the central canal of the spinal cord. Most conspicuous in tanycytes, the protein is concentrated beneath the apical plasma membrane, where cortactin is also present. Moreover, immunoreactivity for actin is particularly strong in the apical cytoplasm of tanycytes and several ependymal cells (this study; and
The results of our immunohistochemical study demonstrate that certain astrocytes located in white matter tracts, obviously corresponding to the particular populations of subpial astrocytes (
Interestingly, our immunochemical and immunohistochemical analyses have revealed that ProSAP1 is also expressed outside the nervous system. In endocrine organs, the protein can be localized to various endocrine cell types of the pituitary and the endocrine pancreas as well as to chromaffin cells of the adrenal medulla (our unpublished observations), where it might be engaged in the microfilament-dependent control of secretory activity. A considerable body of evidence indicates that, in both adenohypophyseal and in pancreatic islet cells, cortical actin filaments participate in the regulation of secretory granule exocytosis (
In the pancreas, the expression of ProSAP1 is not confined to the endocrine compartment. Rather, strong immunoreactivity also becomes visible in the duct system of the exocrine pancreas after incubation with ProSAP1 antibodies. An apical enrichment of ProSAP1 is conspicuous in centroacinar, intercalated duct, and intra/interlobular duct cells. These cells display several ultrastructural similarities, including the presence of a terminal web beneath their apical surface and the formation of many microvilli with a core of axial bundles of microfilaments (
In conclusion, we have established that the expression of ProSAP1, which probably acts as a central organizer of the PSD in neural tissues, is not restricted to neurons. The presence of ProSAP1 in various non-neuronal cells indicates that the function of this protein is neither unique to neurons nor confined to intercellular junctions. It will be interesting to determine all binding partners of ProSAP1 in the former cells and to see whether they differ from the ProSAP1-interacting proteins in neurons. Such an analysis could also give further clues to the targeting mechanisms that specify the polarized distribution of this PDZ protein to discrete cell microdomains, such as the neuronal PSD or the apical cytoplasm of several epithelium-type cells. Therefore, the future elucidation of the function of ProSAP1 in non-neuronal cells not only will provide new insights into the dynamics of their cytoskeleton and its interactions with cell membranes but should also contribute to a comprehensive understanding of the functional microanatomy of the neuronal PSD.
![]() |
Acknowledgments |
---|
Supported by the DFG (SFB426/A1 to EDG, Bo 1718/1-1 to TMB), IZKF and IMF (University of Münster to TMB).
We gratefully acknowledge the skillful technical assistance of H. Böning, S. Fischer, and D. von Mayersbach. We thank Prof E. Ungewickell for providing access to the Nikon Eclipse E800 photomicroscope, and Dr A. Gebert for help with the color prints.
Received for publication August 14, 2000; accepted December 14, 2000.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alper SL, StuartTilley A, Simmons CF, Brown D, Drenckhahn D (1994) The fodrin-ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+K+-ATPase rather than with basolateral anion exchanger AE2. J Clin Invest 93:1430-1438[Medline]
Bitner C, BenjellounTouimi S, Dupouey P (1987) Palisading pattern of subpial astroglial processes in the adult rodent brain: relationship between the glial palisading pattern and the axonal and astroglial organization. Dev Brain Res 37:167-178
Boeckers TM, Kreutz MR, Winter C, Zuschratter W, Smalla K-H, SanmartiVila L, Wex H, Langnaese K, Bockmann J, Garner CC, Gundelfinger ED (1999a) Proline-rich synapse-associated protein-1/cortactin binding protein 1 (ProSAP1/CortBP1) is a PDZ-domain protein highly enriched in the postsynaptic density. J Neurosci 19:6506-6518
Boeckers TM, Winter C, Smalla K-H, Kreutz MR, Bockmann J, Seidenbecher C, Garner CC, Gundelfinger ED (1999b) Proline-rich synapse-associated proteins ProSAP1 and ProSAP2 interact with synaptic proteins of the SAPAP/GKAP family. Biochem Biophys Res Commun 264:247-252[Medline]
Bruni JE (1974) Scanning and transmission electron microscopy of the ependymal lining of the third ventricle. Can J Neurol Sci 1:59-73[Medline]
Carbajal ME, Vitale ML (1997) The cortical actin cytoskeleton of lactotropes as an intracellular target for the control of prolactin secretion. Endocrinology 138:5374-5384
Castellino F, Heuser J, Marchetti S, Bruno B, Luini A (1992) Glucocorticoid stabilization of actin filaments: a possible mechanism for inhibition of corticotropin release. Proc Natl Acad Sci USA 89:3775-3779[Abstract]
Cetin Y, Aunis D, Bader M-F, Galindo E, Jörns A, Bargsten G, Grube D (1993) Chromostatin, a chromogranin A-derived bioactive peptide, is present in human pancreatic insulin (ß) cells. Proc Natl Acad Sci USA 90:2360-2364[Abstract]
Craven SE, Bredt DS (1998) PDZ proteins organize synaptic signaling pathways. Cell 93:495-498[Medline]
Du Y, Weed SA, WenCheng X, Marshall TD, Parsons TJ (1998) Identification of a novel cortactin SH3 domain-binding protein and its localization to growth cones of cultured neurons. Mol Cell Biol 18:5838-5851
Egerbacher M, Böck P (1997) Morphology of the pancreatic duct system in mammals. Microsc Res Tech 37:407-417[Medline]
Ehlers MD (1999) Synapse structure: glutamate receptors connected by the shanks. Curr Biol 9:R848-850[Medline]
Eyigor O, Jennes L (1998) Identification of kainate-preferring glutamate receptor subunit GluR7 mRNA and protein in the rat median eminence. Brain Res 814:231-235[Medline]
Garner CC, Nash J, Huganir RL (2000) PDZ domains in synapse assembly and signalling. Trends Cell Biol 10:274-280[Medline]
GröschelStewart U, Unsicker K, Leonhardt H (1977) Immunohistochemical demonstration of contractile proteins in astrocytes, marginal glial and ependymal cells in rat diencephalons. Cell Tissue Res 180:133-137[Medline]
Horner PJ, Power AE, Kempermann G, Kuhn HG, Palmer TD, Winkler J, Thal LJ, Gage FH (2000) Proliferation and differentiation of progenitor cells throughout the intact adult rat spinal cord. J Neurosci 20:2218-2228
Hosli E, Hosli L (2000) Colocalization of neurotransmitter receptors on astrocytes in explant cultures of rat CNS. Neurochem Int 36:301-311[Medline]
Kawakami S (2000) Glial and neuronal localization of ionotropic glutamate receptor subunit-immunoreactivities in the median eminence of female rats: GluR2/3 and GluR6/7 colocalize with vimentin, not with glial fibrillary acidic protein (GFAP). Brain Res 858:198-204[Medline]
Lessard JL (1988) Two monoclonal antibodies to actin: one muscle selective and one generally reactive. Cell Motil Cytoskel 10:349-362[Medline]
Li G, RunggerBrändle E, Just I, Jonas J-C, Aktories K, Wollheim CB (1994) Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets. Mol Biol Cell 5:1199-1213[Abstract]
Lim S, Naisbitt S, Yoon J, Hwang J-I, Suh P-G, Sheng M, Kim E (1999) Characterization of the Shank family of synaptic proteins. J Biol Chem 274:29510-29518
Liuzzi FJ, Miller RH (1987) Radially oriented astrocytes in the normal adult rat spinal cord. Brain Res 403:385-388[Medline]
Maienschein V, Marxen M, Volknandt W, Zimmermann H (1999) A plethora of presynaptic proteins associated with ATP-storing organelles in cultured astrocytes. Glia 26:233-244[Medline]
Matus A (1999) Postsynaptic actin and neuronal plasticity. Curr Opin Neurobiol 9:561-565[Medline]
Naisbitt S, Kim E, Tu JC, Xiao B, Sala C, Valtschanoff J, Weinberg RJ, Worley PF, Sheng M (1999) Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin. Neuron 23:569-582[Medline]
O'Brien RJ, Lau LF, Huganir RL (1998) Molecular mechanisms of glutamate receptor clustering at excitatory synapses. Curr Opin Neurobiol 8:364-369[Medline]
Orci L, Gabby KH, Malaisse WJ (1972) Pancreatic beta-cell web, its possible role in insulin secretion. Science 175:1128-1130[Medline]
Pabst H, Redecker P (1999) Interstitial glial cells of the gerbil pineal gland display immunoreactivity for the metabotropic glutamate receptors mGluR2/3 and mGluR5. Brain Res 838:60-68[Medline]
Redecker P (1996) Synaptotagmin I, synaptobrevin II, and syntaxin I are coexpressed in rat and gerbil pinealocytes. Cell Tissue Res 283:443-454[Medline]
Redecker P (1998) Developmental pattern of cell type-specific calretinin immunoreactivity in the postnatal gerbil pineal gland. Dev Brain Res 105:43-50
Redecker P (1999) Synaptic-like microvesicles in mammalian pinealocytes. Int Rev Cytol 191:201-255[Medline]
Redecker P, Bargsten G (1993) Synaptophysina common constituent of presumptive secretory microvesicles in the mammalian pinealocyte: a study of rat and gerbil pineal glands. J Neurosci Res 34:79-96[Medline]
Redecker P, Cetin Y, Grube D (1995) Differential distribution of synaptotagmin I and rab3 in the anterior pituitary of four mammalian species. Neuroendocrinology 62:101-110[Medline]
Redecker P, Cetin Y, Korf H-W (1996) Differential immunocytochemical localization of calretinin in the pineal gland of three mammalian species. J Neurocytol 25:9-18[Medline]
Redecker P, Seipelt A, Jörns A, Bargsten G, Grube D (1992) The microanatomy of canine islets of Langerhans: implications for intra-islet regulation. Anat Embryol 185:131-141[Medline]
Senda T, Okabe T, Matsuda M, Fujita H (1994) Quick-freeze, deep-etch visualization of exocytosis in anterior pituitary secretory cells: localization and possible roles of actin and annexin II. Cell Tissue Res 277:51-60[Medline]
Short DB, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ, Milgram SL (1998) An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem 273:19797-19801
Snabes MC, Boyd AE (1982) Increased filamentous actin in islets of Langerhans from fasted hamsters. Biochem Biophys Res Commun 104:207-211[Medline]
Tang FR, Sim MK (1997) Metabotropic glutamate receptor subtype-1 (mGluR1
) immunoreactivity in ependymal cells of the rat caudal medulla oblongata and spinal cord. Neurosci Lett 225:177-180[Medline]
ThomasReetz A, De Camilli P (1994) A role for synaptic vesicles in nonneuronal cells: clues from pancreatic ß cells and from chromaffin cells. FASEB J 8:209-216
Tu JC, Xiao B, Naisbitt S, Yuan JP, Petralia RS, Brakeman P, Doan A, Aakalu VK, Lanahan AA, Sheng M, Worley PF (1999) Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins. Neuron 23:583-592[Medline]
Valentijn K, Valentijn JA, Jamieson JD (1999) Role of actin in regulated exocytosis and compensatory membrane retrieval: insights from an old acquaintance. Biochem Biophys Res Commun 266:652-661[Medline]
Verkhratsky A, Steinhauser C (2000) Ion channels in glial cells. Brain Res Brain Res Rev 32:380-412[Medline]
Wittkowski W (1998) Tanycytes and pituicytes: morphological and functional aspects of neuroglial interaction. Microsc Res Tech 41:29-42[Medline]
Wu H, Montone KT (1998) Cortactin localization in actin-containing adult and fetal tissues. J Histochem Cytochem 46:1189-1191
Wu H, Parsons JT (1993) Cortactin, an 80/ 85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex. J Cell Biol 120:1417-1426[Abstract]
Wu H, Reynolds AB, Kanner SB, Vines RR, Parsons JT (1991) Identification of a novel cytoskeleton-associated pp60src substrate. Mol Cell Biol 11:5113-5124[Medline]
Ziff EB (1997) Enlightening the postsynaptic density. Neuron 19:1163-1174[Medline]