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Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes.

Mundel P, Heid HW, Mundel TM, Krüger M, Reiser J, Kriz W - J. Cell Biol. (1997)

Bottom Line: In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins.The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity.From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Heidelberg, Germany. peter.mundel@urz.uni-heidelberg.de

ABSTRACT
Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9. 27 (mouse). Synaptopodin contains a high amount of proline ( approximately 20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

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Expression of synaptopodin  in a cultured mouse podocyte cell line.  (a) Phase contrast morphology of undifferentiated podocytes. The cobblestone  morphology of undifferentiated podocytes  growing under permissive conditions is  shown. The cells form a monolayer as  they reach confluence. (b) Arborized  podocytes maintained under nonpermissive conditions are very large and flat.  Development of branched processes is  obvious. (c) Induction of synaptopodin  after 4 d at nonpermissive temperature.  While all cells express the podocyte-specific transcription factor WT-1 (green),  cobblestones do not express synaptopodin (red). Currently differentiating cells  show an induction of synaptopodin (arrows). In the center, a differentiated, binucleated, arborized cell is encountered  that expresses high levels of synaptopodin. (d) Expression of synaptopodin in a  differentiated, arborized cell with well  developed processes after 14 d at 37°C.  Synaptopodin is found along the actin  filaments and in focal contacts (arrows).  The association of synaptopodin with actin was confirmed in double labeling experiments (e and f) with rhodamin-conjugated phalloidin. (e) A linear staining  of actin filaments with phalloidin is observed. f shows the punctated distribution of synaptopodin along the same actin  filaments as in e (arrows). (g) Depolymerization of actin filaments with cytochalasin B abolished the linear staining  pattern, and synaptopodin clustered in  the perinuclear cytoplasm. (h) Depolymerization of microtubules with colcemid did not significantly affect the staining  pattern of synaptopodin. Bars, 5 μm.
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Figure 11: Expression of synaptopodin in a cultured mouse podocyte cell line. (a) Phase contrast morphology of undifferentiated podocytes. The cobblestone morphology of undifferentiated podocytes growing under permissive conditions is shown. The cells form a monolayer as they reach confluence. (b) Arborized podocytes maintained under nonpermissive conditions are very large and flat. Development of branched processes is obvious. (c) Induction of synaptopodin after 4 d at nonpermissive temperature. While all cells express the podocyte-specific transcription factor WT-1 (green), cobblestones do not express synaptopodin (red). Currently differentiating cells show an induction of synaptopodin (arrows). In the center, a differentiated, binucleated, arborized cell is encountered that expresses high levels of synaptopodin. (d) Expression of synaptopodin in a differentiated, arborized cell with well developed processes after 14 d at 37°C. Synaptopodin is found along the actin filaments and in focal contacts (arrows). The association of synaptopodin with actin was confirmed in double labeling experiments (e and f) with rhodamin-conjugated phalloidin. (e) A linear staining of actin filaments with phalloidin is observed. f shows the punctated distribution of synaptopodin along the same actin filaments as in e (arrows). (g) Depolymerization of actin filaments with cytochalasin B abolished the linear staining pattern, and synaptopodin clustered in the perinuclear cytoplasm. (h) Depolymerization of microtubules with colcemid did not significantly affect the staining pattern of synaptopodin. Bars, 5 μm.

Mentions: The association of synaptopodin with actin was further analyzed in a conditionally immortal podocyte cell line recently established in our laboratory (Fig 11; Mundel et al., 1997). This cell line can be maintained in two different phenotypes, as undifferentiated cobblestones growing as an epithelial monolayer (Fig. 11 a) and as differentiated, arborized cells equipped with processes (Fig. 11 b); these arborized podocytes always arise by conversion from cobblestones.


Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes.

Mundel P, Heid HW, Mundel TM, Krüger M, Reiser J, Kriz W - J. Cell Biol. (1997)

Expression of synaptopodin  in a cultured mouse podocyte cell line.  (a) Phase contrast morphology of undifferentiated podocytes. The cobblestone  morphology of undifferentiated podocytes  growing under permissive conditions is  shown. The cells form a monolayer as  they reach confluence. (b) Arborized  podocytes maintained under nonpermissive conditions are very large and flat.  Development of branched processes is  obvious. (c) Induction of synaptopodin  after 4 d at nonpermissive temperature.  While all cells express the podocyte-specific transcription factor WT-1 (green),  cobblestones do not express synaptopodin (red). Currently differentiating cells  show an induction of synaptopodin (arrows). In the center, a differentiated, binucleated, arborized cell is encountered  that expresses high levels of synaptopodin. (d) Expression of synaptopodin in a  differentiated, arborized cell with well  developed processes after 14 d at 37°C.  Synaptopodin is found along the actin  filaments and in focal contacts (arrows).  The association of synaptopodin with actin was confirmed in double labeling experiments (e and f) with rhodamin-conjugated phalloidin. (e) A linear staining  of actin filaments with phalloidin is observed. f shows the punctated distribution of synaptopodin along the same actin  filaments as in e (arrows). (g) Depolymerization of actin filaments with cytochalasin B abolished the linear staining  pattern, and synaptopodin clustered in  the perinuclear cytoplasm. (h) Depolymerization of microtubules with colcemid did not significantly affect the staining  pattern of synaptopodin. Bars, 5 μm.
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Figure 11: Expression of synaptopodin in a cultured mouse podocyte cell line. (a) Phase contrast morphology of undifferentiated podocytes. The cobblestone morphology of undifferentiated podocytes growing under permissive conditions is shown. The cells form a monolayer as they reach confluence. (b) Arborized podocytes maintained under nonpermissive conditions are very large and flat. Development of branched processes is obvious. (c) Induction of synaptopodin after 4 d at nonpermissive temperature. While all cells express the podocyte-specific transcription factor WT-1 (green), cobblestones do not express synaptopodin (red). Currently differentiating cells show an induction of synaptopodin (arrows). In the center, a differentiated, binucleated, arborized cell is encountered that expresses high levels of synaptopodin. (d) Expression of synaptopodin in a differentiated, arborized cell with well developed processes after 14 d at 37°C. Synaptopodin is found along the actin filaments and in focal contacts (arrows). The association of synaptopodin with actin was confirmed in double labeling experiments (e and f) with rhodamin-conjugated phalloidin. (e) A linear staining of actin filaments with phalloidin is observed. f shows the punctated distribution of synaptopodin along the same actin filaments as in e (arrows). (g) Depolymerization of actin filaments with cytochalasin B abolished the linear staining pattern, and synaptopodin clustered in the perinuclear cytoplasm. (h) Depolymerization of microtubules with colcemid did not significantly affect the staining pattern of synaptopodin. Bars, 5 μm.
Mentions: The association of synaptopodin with actin was further analyzed in a conditionally immortal podocyte cell line recently established in our laboratory (Fig 11; Mundel et al., 1997). This cell line can be maintained in two different phenotypes, as undifferentiated cobblestones growing as an epithelial monolayer (Fig. 11 a) and as differentiated, arborized cells equipped with processes (Fig. 11 b); these arborized podocytes always arise by conversion from cobblestones.

Bottom Line: In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins.The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity.From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Heidelberg, Germany. peter.mundel@urz.uni-heidelberg.de

ABSTRACT
Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9. 27 (mouse). Synaptopodin contains a high amount of proline ( approximately 20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

Show MeSH