P-Rex2, a Rac-guanine nucleotide exchange factor, is expressed selectively in ribbon synaptic terminals of the mouse retina
© Sherry and Blackburn; licensee BioMed Central Ltd. 2013
Received: 8 December 2012
Accepted: 10 July 2013
Published: 11 July 2013
Phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac Exchanger 2 (P-Rex2) is a guanine nucleotide exchange factor (GEF) that specifically activates Rac GTPases, important regulators of actin cytoskeleton remodeling. P-Rex2 is known to modulate cerebellar Purkinje cell architecture and function, but P-Rex2 expression and function elsewhere in the central nervous system is unclear. To better understand potential roles for P-Rex2 in neuronal cytoskeletal remodeling and function, we performed widefield and confocal microscopy of specimens double immunolabeled for P-Rex2 and cell- and synapse-specific markers in the mouse retina.
P-Rex2 was restricted to the plexiform layers of the retina and colocalized extensively with Vesicular Glutamate Transporter 1 (VGluT1), a specific marker for photoreceptor and bipolar cell terminals. Double labeling for P-Rex2 and peanut agglutinin, a cone terminal marker, confirmed that P-Rex2 was present in both rod and cone terminals. Double labeling with markers for specific bipolar cell types showed that P-Rex2 was present in the terminals of rod bipolar cells and multiple ON- and OFF-cone bipolar cell types. In contrast, P-Rex2 was not expressed in the processes or conventional synapses of amacrine or horizontal cells.
P-Rex2 is associated specifically with the glutamatergic ribbon synaptic terminals of photoreceptors and bipolar cells that transmit visual signals vertically through the retina. The Rac-GEF function of P-Rex2 implies a specific role for P-Rex2 and Rac-GTPases in regulating the actin cytoskeleton in glutamatergic ribbon synaptic terminals of retinal photoreceptors and bipolar cells and appears to be ideally positioned to modulate the adaptive plasticity of these terminals.
KeywordsPhotoreceptor Bipolar cell Cytoskeleton GEF Rac GTPase Actin
The dynamic remodeling of the actin cytoskeleton is critical to the establishment of neuronal architecture and formation of synaptic connections, as well as structural and functional plasticity in response to normal or pathological stimuli. The Rho family of small GTPases, RhoA, Rac, and Cdc42, are key regulators of remodeling of the neuronal actin cytoskeleton and have distinct functions [reviewed in [1–3]]. Rho-family GTPases cycle between an inactive GDP-bound state and an active GTP-bound state, which is regulated by their interactions with a variety of guanine nucleotide exchange factors (GEFs) that promote activation by catalyzing the exchange of GDP for GTP, and GTPase activating proteins (GAPs) that promote inactivation by stimulating the GTPase to hydrolyze GTP to GDP. Therefore, the specific GEFs and GAPs expressed by a neuron and their localization within the cell determine the spatial and temporal activation of Rho-family GTPases and remodeling of the actin cytoskeleton.
Rac1 and Rac3 (also known as Rac1B) are Rho-family GTPases that are expressed in the central nervous system (CNS). Rac1 is important for outgrowth, guidance, and branching of neurites [2, 3]. In contrast, activation of Rac3 appears to inhibit neurite outgrowth and may have a function antagonistic to that of Rac1 . Several GEFs that specifically activate Rac GTPases have been identified and are known to affect development and structure of neuronal processes, consistent with the importance of actin cytoskeleton remodeling to neuronal structure and function (e.g., Tiam 1 and 2, Trio, Kalirin, P-Rex1 and 2) [5–14].
The Phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac Exchanger family of proteins (P-Rex1 and P-Rex2) are GEFs that selectively activate Rac GTPases [15–17]. The P-Rexes are functionally unique in that they require simultaneous signals from PhosphoInositide-3-Kinase (PI3K) and G-protein coupled-receptor (GPCR) pathways for their activation. Thus, they integrate and communicate the coordinated signals in PI3K and GPCR pathways to the actin cytoskeleton through the activation of Rac GTPases [15, 18, 19].
Recent studies specifically implicate the P-Rex family of Rac-GEFs in the regulation of neurite growth and guidance, neuronal architecture, and functional plasticity. P-Rex1 is widely expressed by neurons in the developing central nervous system including neurons in the cerebral cortex and dorsal root ganglion . P-Rex1 localizes to the leading edge of migrating cortical neurons and to neuritic growth cones [11, 13]. Expression of a dominant negative form of P-Rex1 perturbs radial migration of cortical neurons , and knockdown of P-Rex1 in neuronally differentiated PC12 cells inhibits new neurite outgrowth while promoting the differentiation of existing neurites as indicated by enrichment of β tubulin . Expression of P-Rex2 in the brain is much more limited and is most prominent in cerebellar Purkinje cells . Knockout of P-Rex2 leads to aberrant dendritic architecture in cerebellar Purkinje cells and impaired cerebellum-mediated motor function . Impaired P-Rex2 function also disrupts functional synaptic plasticity, interfering with late phase consolidation of long-term potentiation at the parallel fiber-Purkinje cell synapse .
Although it is clear that P-Rex function is important to cell migration, neurite differentiation and architecture during neural development, and to synaptic function in the adult brain, little is known about the distribution and function of P-Rex Rac-GEFs in specific synapses and circuits. To better understand the potential roles that P-Rex2 might play in regulation of the neuronal actin cytoskeleton in adult CNS neurons and circuits, we investigated the distribution of P-Rex2 in the mouse retina, a major model system for studies of neuronal and synaptic development and plasticity. To characterize the expression and localization of P-Rex2 in retinal neurons we analyzed immunolabeling for P-Rex2 in conjunction with well-known markers for specific retinal cell types and synapses at the light and confocal microscopic levels.
Animals and tissue preparation
Studies were performed using retinas from light-adapted adult wildtype C57BL/6 J mice (Jackson Laboratories, Bar Harbor, ME). Mice were kept on a 12-hour light: 12-hour dark cycle, with food and water available ad libitum. Animals were euthanized by rapid cervical dislocation. All animal procedures were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee and conformed to the guidelines of the US Public Health Service and the Institute for Laboratory Animal Research.
Following euthanasia, eyes were enucleated, the cornea was punctured with a needle to facilitate fixation, and the eyes were immersion fixed in 4% paraformaldehyde in 0.1 M cacodylate or 0.1 M phosphate buffered saline (PBS) at pH 7.2 for 30 minutes to 2 hours at 4°C. After fixation, the anterior segment and lens were removed to create an eyecup. Eyecups were rinsed in PBS, cryoprotected in 30% sucrose in PBS, embedded in Optimal Cutting Temperature medium (OCT; Sakura Tissue Tek; VWR, West Chester, PA), and then rapidly frozen in liquid nitrogen. Frozen sections of 10–15 μm thickness were cut on a cryostat, collected on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) and stored at −20°C to −35°C until use.
Antisera, antibodies, and lectins
List of antibodies and lectins used for immunolabeling
Purified Calbindin from bovine kidney
Sigma, St Louis, MO (Catalog # C9848; clone CB-955)
Immunoaffinity purified GAD from rat brain
Developmental Studies Hybridoma Bank, U Iowa, (Catalog # GAD-6; clone GAD-6)
C-terminus peptide sequence from Rat GlyT1
Chemicon, Temecula, CA (Catalog # AB1770)
Microtubules purified from rat brain
Sigma, St Louis, MO (Catalog # M4278; clone HM-1)
Human PKCα, aa. 270-427
B-D Transduction Labs, San Jose, CA (Catalog # 610107; clone 3/PKC)
Invitrogen-Molecular Probes, Carlsbad, CA (Catalog # L21409)
Mouse P-Rex2, aa. 717-799
Dr. Heidi C.E. Welch, The Babraham Institute, Cambridge, England (affinity purified antiserum #78)
Zebrafish hindbrain membranes
Zebrafish International Resource Center, Eugene, OR (Catalog # znp-1; clone znp-1)
Rat hippocampus synaptosomes
Sigma, St Louis, MO (Catalog # S0664; clone HPC-1)
Peptide, aa. 541–560 of rat VGluT1
Chemicon, Temecula, CA (Catalog # AB5905)
Peptide, aa. 493–560 of rat VGluT1
UC Davis/NIH NeuroMab Facility, Davis, CA (Catalog # 75–066; clone N28/9)
Mouse monoclonal anti-Calbindin was raised against purified bovine kidney Calbindin-D-28 K, and does not recognize other members of the EF hand calcium binding protein family. Immunolabeling patterns matched previous reports in the mouse retina . Mouse monoclonal anti-glutamic acid decarboxylase, 65 kDa isoform (GAD-65) was raised against GAD purified from rat brain and recognizes a single band on western blots . GAD-65 immunolabeling matched the pattern reported previously in mouse retina . Goat polyclonal anti-GlyT1 was raised against a C-terminus peptide from rat GlyT1 and produced immunolabeling that matched previous reports in mouse retina [21, 23]. Mouse monoclonal anti-MAP-1 was raised against microtubules purified from rat brain and recognizes a single band corresponding to MAP-1 on western blots of rat brain and does not cross react with MAP-2 . MAP-1 immunolabeling of ganglion cell dendrites in the IPL matches previous reports . Peanut agglutinin (PNA) is a lectin that recognizes β(1–3)-GalNAc carbohydrates and is a specific marker for the extracellular matrix surrounding cone outer segments and the base of cone terminals [26, 27]. Mouse monoclonal anti-PKCα was raised against amino acids 270–427 of human PKCα, recognizes a single 82 kDa band on western blots of rat brain, and specifically immunolabeled rod bipolar cells in the mouse retina as appropriate [21, 28, 29]. Affinity purified rabbit polyclonal anti-P-Rex2 was raised against amino acids 717–799 of mouse P-Rex2 and recognizes a single band on western blots of mouse cerebellar lysates and does not cross-react with P-Rex1 . Mouse monoclonal anti-synaptotagmin 2 was raised against adult zebrafish hindbrain membranes  and recognizes a single 60 kDa band in western blots of mouse cerebellar lysates and synaptosomes identified as synaptotagmin 2 by mass spectrometry . Anti-synaptotagmin 2 labeled horizontal cells, Type 2 OFF-cone bipolar cells, and Type 6 ON-cone bipolar cells as previously reported for mouse retina [28, 31]. Mouse monoclonal anti-syntaxin 1a was raised against rat hippocampal synaptosomes  and recognizes a single 35 kDa band on western blot corresponding to syntaxin 1a . Immunolabeling matched patterns previously reported in retina and selectively labels amacrine cells and their processes and conventional synapses in the inner plexiform layer of the mammalian retina [23, 32, 34]. Two different antibodies directed against VGluT1 were used. The guinea pig polyclonal antibody and the mouse monoclonal antibody both recognize a single 52 kDa band on western blots and do not crossreact with VGluT2. Both VGluT1 antibodies produce immunolabeling of photoreceptor and bipolar cell terminals, matching the previously reported distribution of VGLUT1 in the retina [35, 36].
Methods for immunolabeling of paraffin and frozen sections were as described previously [23, 37]. Paraffin sections were de-paraffinized, and then subjected to antigen retrieval through 100% methanol at −20°C for 20 min, followed by 1% NaBH4, then 0.1 M citrate buffer (pH 6.0) at 85-95°C for 45 min, after which they were exchanged to PBS or Hank’s buffered saline solution (HBSS, pH 7.4) and blocked and processed as described below for frozen sections. Frozen sections were thawed, rehydrated and treated with 1% NaBH4, rinsed, and exchanged to PBS or HBSS. Sections were then incubated in blocking solution (“blocker,” 10% normal goat serum + 5% bovine serum albumin + 1% fish gelatin + 0.5% triton X-100 in PBS or HBSS) for 2 hours at room temperature. Blocker was removed and primary antibody or a cocktail of primary antibodies for double or triple labeling experiments were applied for 1–2 days at 4°C. After removing primary antibody, sections were rinsed in PBS or HBSS, and fluorescent secondary antibody, or a cocktail of secondary antibodies ± PNA for multiple labeling experiments, was applied for 1 to 1.5 hours at room temperature. Sections were rinsed and coverslipped using Prolong Gold + DAPI (Invitrogen-Molecular Probes, Carlsbad, CA). Control sections processed in parallel using normal host sera substituted for primary antisera or by omitting primary antibody showed no labeling. Control sections that received only one primary antibody showed labeling in only the appropriate channel when treated with multiple fluorescent secondary antibodies.
Imaging and analysis
Widefield visualization of immunolabeling was performed using an Olympus BX61WI fluorescence microscope (Olympus America, Center Valley, PA) equipped with a Hamamatsu ORCA-ER camera (Hamamatsu, Bridgewater, NJ). Slidebook software (Intelligent Imaging Innovations, Denver, CO) was used for image acquisition. Confocal microscopy was performed using an Olympus FluoView1000 confocal microscope (Olympus America, Center Valley, PA). Confocal images were acquired using a 60x oil immersion objective lens (NA = 1.42) and Olympus Fluoview software. To prevent bleed-through of signals between fluorescence channels, detector sensitivity and laser power were adjusted and images were collected sequentially in the different fluorescent channels. For preparation of figures, widefield or confocal images were exported to Photoshop (Adobe, Mountain View, CA) and brightness, contrast and threshold were adjusted to highlight specific labeling.
P-Rex2 is expressed specifically in the synaptic layers of the retina
P-Rex2 is expressed specifically in rod and cone terminals in the OPL
P-Rex2 expression in the IPL is restricted to bipolar cell terminals
Together these results indicate that P-Rex2 is localized specifically to the synaptic terminals of bipolar cells in the IPL, but is not present in the synapses or process of amacrine or ganglion cells.
Identification of bipolar cell types expressing P-Rex2
To identify specific types of bipolar cells expressing P-Rex2 in their terminals, we performed double labeling for P-Rex2 in combination with markers for the terminals of specific rod- and cone-driven bipolar cell subtypes.
These studies establish that P-Rex2, a GEF that specifically activates Rac small GTPases, is expressed in a cell- and synapse-specific manner in the retina. P-Rex2 localized to the synaptic terminals of photoreceptor and bipolar cells, as indicated by extensive double labeling with VGluT1 and other bipolar cell terminal markers. In contrast, P-Rex2 was not expressed in horizontal or amacrine cell processes or terminals or in the dendrites of bipolar or ganglion cells, as indicated by the absence of colocalization with an array of cell- and synapse-specific markers, including calbindin, synaptotagmin 2, PKC, GAD-65, GlyT1, and MAP-1 [21, 23, 25, 28, 31, 32, 34]. Thus, P-Rex2 in the retina is expressed specifically in the specialized glutamatergic ribbon synaptic terminals of photoreceptors and bipolar cells that transmit information vertically through the retina, and not the conventional synapses of amacrine cells or the processes of horizontal cells that mediate lateral processing in the IPL and OPL, respectively.
P-Rex2 localized to both rod and cone photoreceptor terminals in the OPL and to both rod and cone bipolar cell terminals in the IPL, indicating that P-Rex2 expression is not restricted specifically to either rod- or cone-driven synaptic pathways. Similarly, P-Rex2 expression is not exclusively associated with either ON or OFF pathways in the retina as P-Rex2 localized to bipolar cell terminals distributed across the ON and OFF sublayers of the IPL. The distribution of P-Rex2-positive bipolar cell terminals throughout the depth of the IPL further indicates that multiple bipolar cell types contained P-Rex 2 in their terminals. Double labeling for P-Rex2 in conjunction with PKC and synaptotagmin 2 positively identified P-Rex2 expression in the terminals of rod bipolar cells and Type 2 and Type 6 cone bipolar cells [21, 28, 31]. These studies also showed that the terminals of many additional bipolar cells also contained P-Rex2. On the basis of depth of stratification and the size characteristics of these terminals, it is clear that P-Rex2 also must be present in the terminals of several additional cone bipolar cell types and potentially could be present in the terminals of all bipolar cell types.
The distribution of P-Rex2 does not precisely match the reported distribution of its effector, Rac1. Rac 1 has been localized to photoreceptor outer segments and undergoes light-induced activation . Activation of Rac1 in the outer segment appears to have a key role in light-induced photoreceptor cell death [40, 41]. P-Rex2 seems unlikely to modulate light induced activation of Rac1 in the outer segment, as our data show little evidence for enrichment of P-Rex2 in photoreceptor outer segments. In contrast, our data suggest that P-Rex2 may be more important in the regulation of Rac1 activity specifically in photoreceptor terminals. Consistent with this notion, Rac1 has been localized to the distal portion of the mouse OPL . Although Rac1 is known to be important for proper development and polarization of Drosophila photoreceptors , conditional knockout of Rac1 from mouse rods does not appear to greatly disrupt the structure or function of mouse rods , although the structural organization and plasticity of photoreceptor terminals in vertebrate photoreceptors lacking Rac1 has not been examined in detail. The expression of Rac1 by cells in the inner retina and Rac1 labeling in the IPL has been reported previously [40, 43], but little is known regarding the cell-specific distribution or activation of Rac1 in the inner retina. The finding that P-Rex2 is selectively localized to bipolar cell terminals suggests that P-Rex2 provides specific regulation of Rac1 activity in those terminals.
The P-Rexes regulate actin cytoskeleton remodeling by activating Rac GTPases. P-Rex activation requires coincident signals via PI3K and G-protein receptor activation [18, 19, 44] and is a key mechanism for the regulation of membrane dynamics and remodeling of cytoskeleton in response to external cues [11, 15, 18, 19, 44, 45]. Diminished P-Rex function in neurons leads to aberrations in growth cone structure, membrane ruffling, neurite outgrowth, and neuritic architecture, resulting in functional deficits and impaired synaptic plasticity [11–13, 20]. It is likely that P-Rex2 serves a similar function in the terminals of photoreceptors and bipolar cells. One attractive possibility is that P-Rex2 may mediate adaptive remodeling of the terminal in response to simultaneous activation of G-protein and PI3K mediated pathways in the terminal. The terminals of photoreceptors and bipolar cells and their synaptic partners undergo significant anatomical remodeling in response to changes in illumination, including the extension and retraction of processes from the terminal itself and rearrangements associated with post-synaptic processes [46–54]. Plasticity of this nature is best known in the retinas of non-mammalian species [48–53], but adaptive structural changes also occur in mammalian photoreceptor and bipolar cell terminals [46, 47, 54]. This structural remodeling is dependent at least in part on the actin cytoskeleton as treatment with cytochalaisin D inhibits remodeling [50, 52], which would be consistent with a role for P-Rex2 in adaptive remodeling.
Another potential function for P-Rex2 is coordination of adaptive remodeling of the synaptic machinery within photoreceptor and bipolar cell terminals, presumably via activation of Rac1 which is known to be present in photoreceptors and other retinal cells [39–41, 43, 55]. For example, synaptic ribbons, and active zones in rod photoreceptor terminals undergo adaptive light-dependent (i.e., activity-dependent) remodeling [56–58]. Ribbon and active zone material is removed in the first few hours after light onset resulting in shortening or disappearance of some synaptic ribbons and active zones, and detachment of other ribbons from the terminal plasma membrane. This remodeling is then reversed in darkness. Synaptic vesicle density can also change with light- or dark-adaptation . The mechanism(s) mediating the movement and remodeling of ribbon and active zone material is currently unknown, but P-Rex2-mediated activation of Rac1 leading to local remodeling of actin within the terminal is a plausible contributor.
P-Rex2 also potentially might modulate functional plasticity at photoreceptor and bipolar cell terminals via the regulation of receptors at the surface of the terminal. Double knockout of P-Rex1 and 2 interferes specifically with late phase consolidation of long-term potentiation at the parallel fiber to Purkinje cell synapse in the cerebellum, most likely due to the failure of the synapse to consolidate changes in AMPA receptor density in the absence of P-Rex . It is unlikely that P-Rex2 would modulate functional plasticity at photoreceptor or bipolar cell terminals by direct modulation of transmitter release, as synaptic vesicle exocytosis is not actin-dependent. However, P-Rex2 activation potentially could affect recycling and trafficking of synaptic vesicles in the reserve pool, which are tethered to the actin cytoskeleton by the synaptic vesicle protein synapsin in conventional synapses [60–62]. However, it is not clear whether P-Rex2 might modulate synaptic vesicle interactions with the actin cytoskeleton in photoreceptor and bipolar terminals, which lack synapsins .
While the signals that regulate P-Rex2 activity in the terminals of photoreceptors and bipolar cells are not known, P-Rex2 activation requires coincident signaling via PI3K and G-protein-coupled receptor mechanisms [18, 19, 44]. Furthermore, the available evidence suggests that activated P-Rexes translocate to spatially restricted domains of the plasma membrane in order to activate Rac GTPases [19, 45]. Thus, P-Rex2 activation would appear to be ideally suited to tight spatial and temporal regulation of Rac GTPase activation in photoreceptor and bipolar cell terminals in response to very specific combinations of external signals. Although the precise signals that activate P-Rex2 in photoreceptor and bipolar cell terminals are not known, defects in phosphoinositide signaling disrupt the structure, maintenance and function of photoreceptor terminals [64, 65] and photoreceptors and bipolar cells possess a variety of G-protein coupled receptors that might contribute to activation of P-Rex2. A challenge for the future will be to define the precise functional role of P-Rex2 in ribbon synaptic terminals and the signals regulating its function.
These studies indicate that P-Rex2 is associated specifically with the ribbon synaptic terminals of photoreceptors and bipolar cells in the retina. These synapses are highly specialized for rapid glutamate release and serve to transmit visual signals vertically through the retina. The selective expression of P-Rex2 at ribbon synapses suggests a role for P-Rex2 associated specifically with ribbon synapse function that is not shared with the conventional synapses of the retina, which typically utilize inhibitory neurotransmitters and do not show labeling for P-Rex2. Because P-Rex2 is a Rac-GEF, it is likely that it regulates the activity of Rac-GTPases and the actin cytoskeleton in glutamatergic ribbon synaptic terminals of retinal photoreceptors and bipolar cells. It is highly unlikely that actin is directly involved in glutamate release from ribbon synapses, but P-Rex2 would be ideally positioned to modulate rearrangements of the actin cytoskeleton that must accompany plastic changes in the shape and conformation of photoreceptor and bipolar terminals associated with light and dark adaptation.
Central nervous system
GTPase activating protein
Glutamic acid decarboxylase, 65 kDa isoform
Ganglion Cell Layer
Guanine nucleotide exchange factor
Glycine transporter 1
G-Protein coupled receptor
Inner nuclear layer
Inner plexiform layer
Microtubule-associated protein 1
Outer nuclear layer
Outer plexiform layer
Protein Kinase Cα
Phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac Exchanger 2
Vesicular glutamate transporter 1.
These studies were supported by OCAST grant# HR08-149S (to DMS); P20RR017703 and P30EY012190 from NIH (to University of Oklahoma Health Sciences Center). We thank Dr. Heidi Welch for the gift of P-Rex2 antiserum and Dr. Jody Summers for use of the confocal microscope. The synaptotagmin 2 monoclonal antibody generated by Trevarrow et al.  was obtained from the Zebrafish International Resource Center, which is supported by grant P40 RR012546 from the NIH-NCRR. The GAD-65 monoclonal antibody developed by Chang and Gottlieb  was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. The monoclonal antibody against VGluT1 (clone N28/9) was developed by and/or obtained from the UC Davis/NIH NeuroMab Facility, supported by NIH grant U24NS050606 and maintained by the Department of Neurobiology, Physiology and Behavior, College of Biological Sciences, University of California, Davis, CA 95616.
- Luo L: Rho GTPases in neuronal morphogenesis. Nat Rev Neurosci. 2000, 1: 173-180. 10.1038/35044547.View ArticlePubMedGoogle Scholar
- Govek EE, Newey SE, Van Aelst L: The role of the Rho GTPases in neuronal development. Genes Dev. 2005, 19: 1-49. 10.1101/gad.1256405.View ArticlePubMedGoogle Scholar
- De Curtis I: Functions of Rac GTPases during neuronal development. Dev Neurosci. 2008, 30: 47-58. 10.1159/000109851.View ArticlePubMedGoogle Scholar
- Hajdo-Milasinovic A, Ellenbroek SI, Van Es S, van der Vaart B, Collard JG: Rac1 and Rac3 have opposing functions in cell adhesion and differentiation of neuronal cells. J Cell Sci. 2007, 120: 555-566. 10.1242/jcs.03364.View ArticlePubMedGoogle Scholar
- Van Leeuwen FN, Kain HE, Kammen RA, Michiels F, Kranenburg OW, Collard JG: The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; opposing roles for the small GTPases Rac and Rho. J Cell Biol. 1997, 139: 797-807. 10.1083/jcb.139.3.797.View ArticleGoogle Scholar
- Bateman J, Shu H, Van Vactor D: The guanine nucleotide exchange factor trio mediates axonal development in the Drosophila embryo. Neuron. 2000, 26: 93-106. 10.1016/S0896-6273(00)81141-1.View ArticlePubMedGoogle Scholar
- Penzes P, Johnson RC, Kambampati V, Mains RE, Eipper BA: Distinct roles for the two Rho GDP/GTP exchange factor domains of kalirin in regulation of neurite growth and neuronal morphology. J Neurosci. 2001, 21: 8426-8433.PubMedGoogle Scholar
- Matsuo N, Hoshino M, Yoshizawa M, Nabeshima Y: Characterization of STEF, a guanine nucleotide exchange factor for Rac1, required for neurite growth. J Biol Chem. 2002, 277: 2860-2868. 10.1074/jbc.M106186200.View ArticlePubMedGoogle Scholar
- Tanaka M, Ohashi R, Nakamura R, Shinmura K, Kamo T, Sakai R, Sugimura H: Tiam1 mediates neurite outgrowth induced by ephrin-B1 and EphA2. EMBO J. 2004, 23: 1075-1088. 10.1038/sj.emboj.7600128.PubMed CentralView ArticlePubMedGoogle Scholar
- Tudor EL, Perkinton MS, Schmidt A, Ackerley S, Brownlees J, Jacobsen NJ, Byers HL, Ward M, Hall A, Leigh PN, Shaw CE, McLaughlin DM, Miller CC: ALS2/ALSIN regulates RAC-PAK signalling and neurite outgrowth. J Biol Chem. 2005, 280: 34735-34740. 10.1074/jbc.M506216200.View ArticlePubMedGoogle Scholar
- Yoshizawa M, Kawauchi T, Sone M, Nishimura YV, Terao M, Chihama K, Nabeshima YJ, Hoshino M: Involvement of a Rac activator, P-Rex1, in neurotrophin-derived signaling and neuronal migration. J Neurosci. 2005, 25: 4406-4419. 10.1523/JNEUROSCI.4955-04.2005.View ArticlePubMedGoogle Scholar
- Donald S, Humby T, Fyfe I, Segonds-Pichon A, Walker SA, Andrews SR, Coadwell WJ, Emson P, Wilkinson LS, Welch HC: P-Rex2 regulates Purkinje cell dendrite morphology and motor coordination. Proc Natl Acad Sci USA. 2008, 105: 4483-4488. 10.1073/pnas.0712324105.PubMed CentralView ArticlePubMedGoogle Scholar
- Waters JE, Astle MV, Ooms LM, Balamatsias D, Gurung R, Mitchell CA: P-Rex1 – a multidomain protein that regulates neurite differentiation. J Cell Sci. 2008, 121: 2892-2903. 10.1242/jcs.030353.View ArticlePubMedGoogle Scholar
- Cahill ME, Xie Z, Day M, Photowala H, Barbolina MV, Miller CA, Weiss C, Radulovic J, Sweatt JD, Disterhoft JF, Surmeier DJ, Penzes P: Kalirin regulates cortical spine morphogenesis and disease-related behavioral phenotypes. Proc Natl Acad Sci USA. 2009, 106: 13058-13063. 10.1073/pnas.0904636106.PubMed CentralView ArticlePubMedGoogle Scholar
- Welch HC, Condliffe AM, Milne LJ, Ferguson GJ, Hill K, Webb LM, Okkenhaug K, Coadwell WJ, Andrews SR, Thelen M, Jones GE, Hawkins PT, Stephens LR: P-Rex1 regulates neutrophil function. Current Biol. 2005, 15: 1867-1873. 10.1016/j.cub.2005.09.050.View ArticleGoogle Scholar
- Donald S, Hill K, Lecureuil C, Barnouin R, Krugmann S, John Coadwell W, Andrews SR, Walker SA, Hawkins PT, Stephens LR, Welch HC: P-Rex2, a new guanine-nucleotide exchange factor for Rac. FEBS Let. 2004, 572: 172-176. 10.1016/j.febslet.2004.06.096.View ArticleGoogle Scholar
- Rosenfeldt H, Vázquez-Prado J, Gutkind JS: P-REX2, a novel PI-3-kinase sensitive Rac exchange factor. FEBS Lett. 2004, 572: 167-171. 10.1016/j.febslet.2004.06.097.View ArticlePubMedGoogle Scholar
- Hill K, Krugmann S, Andrews SR, Coadwell WJ, Finan P, Welch HCE, Hawkins PT, Stephens LR: Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-triphosphate and Gβγ Subunits. J Biol Chem. 2005, 280: 4166-4173.View ArticlePubMedGoogle Scholar
- Barber MA, Donald S, Thelen S, Anderson KE, Thelen M, Welch HC: Membrane translocation of P-Rex1 is mediated by G protein βγ subunits and phosphoinositide 3-kinase. J Biol Chem. 2007, 282: 29967-29976. 10.1074/jbc.M701877200.View ArticlePubMedGoogle Scholar
- Jackson C, Welch HC, Bellamy TC: Control of cerebellar long-term potentiation by P-Rex-family guanine-nucleotide exchange factors and phosphoinositide 3-kinase. PLoS One. 2010, 5: e11962-10.1371/journal.pone.0011962.PubMed CentralView ArticlePubMedGoogle Scholar
- Haverkamp S, Wässle H: Immunocytochemical analysis of the mouse retina. J Comp Neurol. 2000, 424: 1-23. 10.1002/1096-9861(20000814)424:1<1::AID-CNE1>3.0.CO;2-V.View ArticlePubMedGoogle Scholar
- Chang YC, Gottlieb DI: Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase. J Neurosci. 1988, 8: 2123-2130.PubMedGoogle Scholar
- Sherry DM, Mitchell R, Standifer KM, Du Plessis B: Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina. BMC Neurosci. 2006, 7: 54-10.1186/1471-2202-7-54.PubMed CentralView ArticlePubMedGoogle Scholar
- Huber G, Matus A: Differences in the cellular distributions of two microtubule-associated proteins, MAP1 and MAP2, in rat brain. J Neurosci. 1984, 4: 151-160.PubMedGoogle Scholar
- Tucker RP, Matus AI: Microtubule-associated proteins characteristic of embryonic brain are found in the adult mammalian retina. Dev Biol. 1988, 130: 423-434. 10.1016/0012-1606(88)90338-7.View ArticlePubMedGoogle Scholar
- Blanks JC, Johnson LV: Selective lectin binding of the developing mouse retina. J Comp Neurol. 1983, 221: 31-41. 10.1002/cne.902210103.View ArticlePubMedGoogle Scholar
- Blanks JC, Johnson LV: Specific binding of peanut lectin to a class of retinal photoreceptor cells. A species comparison. Invest Ophthalmol Vis Sci. 1984, 25: 546-557.PubMedGoogle Scholar
- Wässle H, Puller C, Muller F, Haverkamp S: Cone contacts, mosaics, and territories of bipolar cells in the mouse retina. J Neurosci. 2009, 29: 106-117. 10.1523/JNEUROSCI.4442-08.2009.View ArticlePubMedGoogle Scholar
- Negishi K, Kato S, Teranishi T: Dopamine cells and rod bipolar cells contain protein kinase C-like immunoreactivity in some vertebrate retinas. Neurosci Lett. 1999, 94: 247-252.View ArticleGoogle Scholar
- Trevarrow B, Marks DL, Kimmel CB: Organization of hindbrain segments in the zebrafish embryo. Neuron. 1990, 4: 669-679. 10.1016/0896-6273(90)90194-K.View ArticlePubMedGoogle Scholar
- Fox MA, Sanes JR: Synaptotagmin I and II are present in distinct subsets of central synapses. J Comp Neurol. 2007, 503: 280-296. 10.1002/cne.21381.View ArticlePubMedGoogle Scholar
- Barnstable CJ, Hofstein R, Akagawa K: A marker of early amacrine cell development in rat retina. Brain Res. 1985, 352: 286-290.View ArticlePubMedGoogle Scholar
- Inoue A, Obata K, Akagawa K: Cloning and sequence analysis of cDNA for a neuronal cell membrane antigen HPC-1. J Biol Chem. 1992, 267: 10613-10619.PubMedGoogle Scholar
- Morgans CW, Brandstätter JH, Kellerman J, Betz H, Wässle H: A SNARE complex containing syntaxin 3 is present in ribbon synapses of the retina. J Neurosci. 1996, 16: 6713-6721.PubMedGoogle Scholar
- Johnson J, Tian N, Caywood MS, Reimer RJ, Edwards RH, Copenhagen DR: Vesicular neurotransmitter expression in developing postnatal rodent retina: GABA and glycine precede glutamate. J Neurosci. 2003, 23: 518-529.PubMedGoogle Scholar
- Sherry DM, Wang MM, Bates J, Frishman LJ: Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits. J Comp Neurol. 2003, 465: 480-498. 10.1002/cne.10838.View ArticlePubMedGoogle Scholar
- Sherry DM, Murray A, Hoffhines A, Kanan Y, Arbogast KL, Fleisler SJ, Burns ME, Moore KL, Al-Ubaidi MR: Lack of protein tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy. Eur J Neurosci. 2010, 32: 1461-1472. 10.1111/j.1460-9568.2010.07431.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Ghosh KK, Bujan S, Haverkamp S, Feigenspan A, Wässle H: Types of bipolar cells in the mouse retina. J Comp Neurol. 2004, 469: 70-82. 10.1002/cne.10985.View ArticlePubMedGoogle Scholar
- Balasubramanian N, Slepak VZ: Light-mediated activation of Rac-1 in photoreceptor outer segments. Curr Biol. 2003, 13: 1306-1310. 10.1016/S0960-9822(03)00511-6.View ArticlePubMedGoogle Scholar
- Belmonte MA, Santos MF, Kihara AH, Yan CYI, Hamassaki DE: Light-induced photoreceptor degeneration in the mouse involves activation of the small GTPase Rac1. Invest Ophthalmol Vis Sci. 2006, 47: 1193-1200. 10.1167/iovs.05-0446.View ArticlePubMedGoogle Scholar
- Haruta M, Bush RA, Kjellstrom S, Vijaysarathy C, Zeng Y, Le Y-Z, Sieving PA: Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function. Proc Nat Acad Sci USA. 2009, 106: 9397-9402. 10.1073/pnas.0808940106.PubMed CentralView ArticlePubMedGoogle Scholar
- Chang HY, Ready DF: Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1. Science. 2000, 290: 1978-1980. 10.1126/science.290.5498.1978.View ArticlePubMedGoogle Scholar
- Mitchell DC, Bryan BA, Liu JP, Liu WB, Zhang L, Qu J, Zhou X, Liu M, Li DW: Developmental expression of three small GTPases in the mouse eye. Molec Vis. 2007, 13: 1144-1153.PubMed CentralPubMedGoogle Scholar
- Welch HC, Coadwell WJ, Ellson CD, Ferguson GJ, Andrews SR, Erdjument-Bromage H, Tempst P, Hawkins PT, Stephens LR: P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine nucleotide exchange factor for Rac. Cell. 2002, 108: 809-821. 10.1016/S0092-8674(02)00663-3.View ArticlePubMedGoogle Scholar
- Zhao T, Nalbant P, Hoshino M, Dong X, Wu D, Bokoch GM: Signaling requirements for translocation of P-Rex1, a key Rac2 exchange factor involved in chemoattractant stimulated human neutrophil function. J Leukocyte Biol. 2007, 81: 1127-1136. 10.1189/jlb.0406251.View ArticlePubMedGoogle Scholar
- Brandon C, Lam DM: The ultrastructure of rat rod synaptic terminals: effects of dark-adaptation. J Comp Neurol. 1983, 217: 167-175. 10.1002/cne.902170205.View ArticlePubMedGoogle Scholar
- McCartney MD, Dickson DH: Morphometric analysis of circadian variations in the retinal photoreceptor synaptic terminals of the adult and fetal guinea pig. Am J Anat. 1986, 176: 1-17. 10.1002/aja.1001760102.View ArticlePubMedGoogle Scholar
- Yazulla S, Studholme KM: Glycine-receptor immunoreactivity in retinal bipolar cells is postsynaptic to glycinergic and GABAergic amacrine cell synapses. J Comp Neurol. 1991, 310: 11-20. 10.1002/cne.903100104.View ArticlePubMedGoogle Scholar
- Yazulla S, Studholme KM: Light-dependent plasticity of the synaptic terminals of Mb bipolar cells in goldfish retina. J Comp Neurol. 1992, 320: 521-530. 10.1002/cne.903200409.View ArticlePubMedGoogle Scholar
- Schmitz Y, Kohler K: Spinule formation in the fish retina: is there an involvement of actin and tubulin? An electron microscopic immunogold study. J Neurocytol. 1993, 22: 205-214. 10.1007/BF01246359.View ArticlePubMedGoogle Scholar
- Wagner HJ, Djamgoz MB: Spinules: a case for retinal synaptic plasticity. Trends Neurosci. 1993, 16: 201-206. 10.1016/0166-2236(93)90155-F.View ArticlePubMedGoogle Scholar
- Weiler R, Janssen-Bienhold U: Spinule-type neurite outgrowth from horizontal cells during light adaptation in the carp retina: an actin-dependent process. J Neurocytol. 1993, 22: 129-133. 10.1007/BF01181576.View ArticlePubMedGoogle Scholar
- Behrens UD, Wagner HJ: Adaptation-dependent changes of bipolar cell terminals in fish retina: effects on overall morphology and spinule formation in Ma and Mb cells. Vision Res. 1996, 36: 3901-3911. 10.1016/S0042-6989(96)00139-3.View ArticlePubMedGoogle Scholar
- Behrens UD, Kasten P, Wagner HJ: Adaptation-dependent plasticity of rod bipolar cell axon terminal morphology in the rat retina. Cell Tissue Res. 1998, 294: 243-251. 10.1007/s004410051174.View ArticlePubMedGoogle Scholar
- Santos-Bredariol AS, Santos MF, Hamassaki-Britto DE: Distribution of the small molecular weight GTP-binding proteins Rac1, Cdc42, RhoA and RhoB in the developing chick retina. J Neurocytol. 2002, 31: 149-159. 10.1023/A:1023997506760.View ArticlePubMedGoogle Scholar
- Adly MA, Spiwoks–Becker I, Vollrath L: Ultrastructural changes of photoreceptor synaptic ribbons in relation to time of day and illumination. Invest Ophthalmol Vis Sci. 1999, 40: 2165-2172.PubMedGoogle Scholar
- Spiwoks-Becker I, Glas M, Lasarzik I, Vollrath L: Mouse photoreceptor synaptic ribbons lose and regain material in response to illumination changes. Eur J Neurosci. 2004, 19: 1559-1571. 10.1111/j.1460-9568.2004.03198.x.View ArticlePubMedGoogle Scholar
- Regus-Leidig H, Specht D, tom Dieck S, Brandstätter JH: Stability of active zone components at the photoreceptor ribbon complex. Molec Vis. 2010, 16: 2690-2700.PubMed CentralPubMedGoogle Scholar
- Yazulla S, Studholme KM: Light adaptation affects synaptic vesicle density but not the distribution of GABAA receptors in goldfish photoreceptor terminals. Microsc Res Tech. 1997, 36: 43-56. 10.1002/(SICI)1097-0029(19970101)36:1<43::AID-JEMT4>3.0.CO;2-#.View ArticlePubMedGoogle Scholar
- Petrucci TC, Morrow JS: Synapsin I: and actin-bundling protein under phosphorylation control. J Cell Biol. 1987, 105: 1355-1363. 10.1083/jcb.105.3.1355.View ArticlePubMedGoogle Scholar
- Benfenati F, Valtorta F, Chieregatti E, Greengard P: Interaction of free and synaptic vesicle-bound synapsin I with F-actin. Neuron. 1992, 8: 377-386. 10.1016/0896-6273(92)90303-U.View ArticlePubMedGoogle Scholar
- Hilfiker S, Benfenati F, Doussau F, Nairn AC, Czernik AJ, Augustine GJ, Greengard P: Structural domains involved in the regulation of transmitter release by synapsins. J Neurosci. 2005, 25: 2658-2669. 10.1523/JNEUROSCI.4278-04.2005.View ArticlePubMedGoogle Scholar
- Mandell JW, Townes-Anderson E, Czernik AJ, Cameron R, Greengard P, De Camilli P: Synapsins in the vertebrate retina: absence from ribbon synapses and heterogeneous distribution among conventional synapses. Neuron. 1990, 5: 19-33. 10.1016/0896-6273(90)90030-J.View ArticlePubMedGoogle Scholar
- Schmitz F, Drenckhahn D: Li+-induced structural changes of synaptic ribbons are related to the phosphoinositide metabolism in photoreceptor synapses. Brain Res. 1993, 604: 142-148. 10.1016/0006-8993(93)90360-Y.View ArticlePubMedGoogle Scholar
- Ivanovich I, Anderson RE, Le YZ, Fliesler SJ, Sherry DM, Rajala RVS: Deletion of the p85α regulatory subunit of phosphoinositide 3-kinase in cone photoreceptor cells results in cone photoreceptor degeneration. Invest Ophthalmol Vis Sci. 2011, 52: 3775-3783. 10.1167/iovs.10-7139.View ArticleGoogle Scholar