Christine Richardson

The plant cytoskeleton, NET3C, and VAP27 mediate the link between the plasma membrane and endoplasmic reticulum

The cortical endoplasmic reticulum (ER) network in plants is a highly dynamic structure, and it contacts the plasma membrane (PM) at ER-PM anchor/contact sites. These sites are known to be essential for communication between the ER and PM for lipid transport, calcium influx, and ER morphology in mammalian and fungal cells. The nature of these contact sites is unknown in plants, and here, we have identified a complex that forms this bridge. This complex includes (1) NET3C, which belongs to a plant-specific superfamily (NET) of actin-binding proteins, (2) VAP27, a plant homolog of the yeast Scs2 ER-PM contact site protein, and (3) the actin and microtubule networks. We demonstrate that NET3C and VAP27 localize to puncta at the PM and that NET3C and VAP27 form homodimers/oligomers and together form complexes with actin and microtubules. We show that F-actin modulates the turnover of NET3C at these puncta and microtubules regulate the exchange of VAP27 at the same sites. Based on these data, we propose a model for the structure of the plant ER-PM contact sites.

Autogenic modulation of mechanoreceptor excitability by glutamate release from synaptic‐like vesicles: evidence from the rat muscle spindle primary sensory ending

Fifty‐nanometre diameter, clear, synaptic‐like vesicles (SLVs) are found in primary mechanosensory nerve terminals of vertebrate and invertebrate animals. We have investigated their role in mechanosensory function using the muscle spindle primary endings of rat Ia afferents as a model. Uptake and release of the synaptic vesicle marker FM1‐43 indicated that SLVs recycle like synaptic vesicles and do so in a Ca2+‐sensitive manner. Mechanical stimulation increased SLV recycling, increasing both dye uptake and release. Immunogold/electronmicroscopy showed that, like the central synaptic endings, Ia peripheral endings are enriched with glutamate. Moreover, exogenous glutamate enhanced stretch‐induced Ia excitability. Enhanced excitability persisted in the presence of antagonists to the commonest ionotropic and metabotropic glutamate receptors (kynurenate, MCPG, CPPG and MAP4). However, excitation by glutamate was abolished by (R,S)‐3,5‐dihydroxyphenylglycine (DHPG), and rather more effectively by (2R,1′‐S,2′‐R,3′‐S)‐2‐(2′‐carboxy‐3′‐phenylcyclopropyl) glycine (PCCG‐13). PCCG‐13 also significantly reduced stretch‐activated excitability in the absence of exogenous glutamate. These data indicate that SLVs recycle at rest, releasing glutamate, and that mechanical activity increases this process. The blockade with DHPG and PCCG‐13 suggests that endogenous glutamate release acts, at least in part, through the recently described phospholipase D‐linked metabotropic Glu receptor to maintain the excitability of the sensory endings.

Plant VAP27 proteins: domain characterization, intracellular localization and role in plant development

The endoplasmic reticulum (ER) is connected to the plasma membrane (PM) through the plant-specific NETWORKED protein, NET3C, and phylogenetically conserved vesicle-associated membrane protein-associated proteins (VAPs). Ten VAP homologues (VAP27-1 to 27-10) can be identified in the Arabidopsis genome and can be divided into three clades. Representative members from each clade were tagged with fluorescent protein and expressed in Nicotiana benthamiana. Proteins from clades I and III localized to the ER as well as to ER/PM contact sites (EPCSs), whereas proteins from clade II were found only at the PM. Some of the VAP27-labelled EPCSs localized to plasmodesmata, and we show that the mobility of VAP27 at EPCSs is influenced by the cell wall. EPCSs closely associate with the cytoskeleton, but their structure is unaffected when the cytoskeleton is removed. VAP27-labelled EPCSs are found in most cell types in Arabidopsis, with the exception of cells in early trichome development. Arabidopsis plants expressing VAP27-GFP fusions exhibit pleiotropic phenotypes, including defects in root hair morphogenesis. A similar effect is also observed in plants expressing VAP27 RNAi. Taken together, these data indicate that VAP27 proteins used at EPCSs are essential for normal ER-cytoskeleton interaction and for plant development.

Cerebellar Golgi, Purkinje, and basket cells have reduced γ-aminobutyric acid immunoreactivity in stargazer mutant mice

The stargazer mutant mouse has characteristic ataxia and head‐tossing traits coupled with a severe impairment in the acquisition of classical eye‐blink conditioning (Qiao et al. [ 1996 ] J. Neurosci. 16:640–648; Qiao et al. [ 1998 ] J. Neurosci. 18:6990–6999). These phenotypes are thought to be cerebellar mediated and have been attributed to the specific reduction in brain‐derived neurotrophic factor (BDNF). The granule cells in the cerebellum of the stargazer mouse exhibit a near‐total and exclusive ablation of BDNF mRNA expression and a consequent defect in TrkB receptor signalling. To investigate whether the stargazer mutation and lack of availability of BDNF in the granule cells compromise the phenotype of the cerebellar inhibitory neurons, specifically their immunoreactivity for γ‐aminobutyric acid (GABA); the levels of GABA neurotransmitter expressed in Golgi, Purkinje, and basket cells; and the density of their synaptic contacts were compared in stargazer and wild‐type controls using electron microscopy and quantitative immunogold labelling. The data presented in this study clearly show that, in the spontaneous ataxic mutant mouse stargazer, the cerebellar inhibitory neurons have significantly reduced levels of GABA immunoreactivity indicative of a significant decrease in their GABA content compared with wild‐type controls. Furthermore, the density of inhibitory synapses between Golgi interneurons and granule cells and also between basket and Purkinje cells in stargazer mutants is reduced to approximately half that in wild‐type controls. Whether this reduction in GABA content and inhibitory synapse density is directly attributable to the lack of BDNF in the cerebellum of the stargazer mutant is yet to be proved. J. Comp. Neurol. 453:85–99, 2002.

Phenotype of cerebellar glutamatergic neurons is altered in stargazer mutant mice lacking brain-derived neurotrophic factor mRNA expression.

Brain‐derived neurotrophic factor (BDNF) influences neuronal survival, differentiation, and maturation. More recently, its role in synapse formation and plasticity has also emerged. In the cerebellum of the spontaneous recessive mutant mouse stargazer (stg) there is a specific and pronounced deficit in BDNF mRNA expression. BDNF protein levels in the cerebellum as a whole are reduced by 70%, while in the granule cells (GCs) there is a selective and near total reduction in BDNF mRNA expression. Recently, we published data demonstrating that inhibitory neurons in the cerebella of stgs have significantly reduced levels (∼50%) of γ‐aminobutyric acid (GABA) and fewer, smaller inhibitory synapses compared to wildtype (WT) controls. Our current investigations indicate that the stargazer mutation has an even more pronounced effect on the phenotype of glutamatergic neurons in the cerebellum. There is a profound decrease in the levels of glutamate‐immunoreactivity (up to 77%) in stg compared to WT controls. The distribution profile of presynaptic vesicles is also markedly different: stgs have proportionally fewer docked vesicles and fewer vesicles located adjacent to the active zone ready to dock than WTs. Furthermore, the thickness of the postsynaptic density (PSD) at mossy fiber‐granule cell (MF‐GC) and parallel fiber‐Purkinje cell (PF‐PC) synapses is severely reduced (up to 33% less than WT controls). The number and length of excitatory synapses, however, appear to be relatively unchanged. It is possible that at least some of theses changes in phenotype are directly attributable to the lack of BDNF in the cerebellum of the stg mutant. J. Comp. Neurol. 481:145–159, 2005.

Liposome-doped hydrogel for implantable tissue

The fabrication, characterisation and optimisation of a PVA hydrogel loaded with trehalose-protected uni-lamellar liposomes produced by freeze thaw cycles is described. By avoiding chemical and heat triggers for cross-linking of the polymer network, for the first time a system has been prepared whose pH is maintained at the physiological value, making it an excellent candidate for implantable tissue applications and in vitro studies of/mimicking biological functions. A combination of cyro-SEM (Scanning Electron Microscopy), time resolved fluorescence spectroscopy, TEM (Transmission Emission Microscopy), DSC (Differential Scanning Calorimetry) and Electrochemical Impedance Spectroscopy (EIS) confirm that the liposomes are thermodynamically stable within the hydrogels.

Repo-Man/PP1 regulates heterochromatin formation in interphase

Repo-Man is a protein phosphatase 1 (PP1) targeting subunit that regulates mitotic progression and chromatin remodelling. After mitosis, Repo-Man/PP1 remains associated with chromatin but its function in interphase is not known. Here we show that Repo-Man, via Nup153, is enriched on condensed chromatin at the nuclear periphery and at the edge of the nucleopore basket. Repo-Man/PP1 regulates the formation of heterochromatin, dephosphorylates H3S28 and it is necessary and sufficient for heterochromatin protein 1 binding and H3K27me3 recruitment. Using a novel proteogenomic approach, we show that Repo-Man is enriched at subtelomeric regions together with H2AZ and H3.3 and that depletion of Repo-Man alters the peripheral localization of a subset of these regions and alleviates repression of some polycomb telomeric genes. This study shows a role for a mitotic phosphatase in the regulation of the epigenetic landscape and gene expression in interphase.

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells

Summary While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells.

A rim-and-spoke hypothesis to explain the biomechanical roles for cytoplasmic intermediate filament networks

ABSTRACT Textbook images of keratin intermediate filament (IF) networks in epithelial cells and the functional compromization of the epidermis by keratin mutations promulgate a mechanical role for this important cytoskeletal component. In stratified epithelia, keratin filaments form prominent radial spokes that are focused onto cell-cell contact sites, i.e. the desmosomes. In this Hypothesis, we draw attention to a subset of keratin filaments that are apposed to the plasma membrane. They form a rim of filaments interconnecting the desmosomes in a circumferential network. We hypothesize that they are part of a rim-and-spoke arrangement of IFs in epithelia. From our review of the literature, we extend this functional role for the subplasmalemmal rim of IFs to any cell, in which plasma membrane support is required, provided these filaments connect directly or indirectly to the plasma membrane. Furthermore, cytoplasmic IF networks physically link the outer nuclear and plasma membranes, but their participation in mechanotransduction processes remain largely unconsidered. Therefore, we also discuss the potential biomechanical and mechanosensory role(s) of the cytoplasmic IF network in terms of such a rim (i.e. subplasmalemmal)-and-spoke arrangement for cytoplasmic IF networks. Summary: The cytoplasmic intermediate filament comprises a rim and spoke arrangement supporting the plasma membrane and connecting to the nucleus, to deliver their mechanosensory functions.

A novel function for the MAP kinase SMA-5 in intestinal tube stability.

In vivo evidence links SMA-5 to the maintenance of the apical domain in the Caenorhabditis elegans intestine. sma-5 mutations induce morphological and biochemical changes of the intermediate filament system, demonstrating the close relationship between posttranslational modification and structural integrity of the evolutionarily conserved intestinal cytoskeleton.