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We previously reported that the membrane skeletal protein 4.1G in the peripheral nervous system transports membrane palmitoylated protein 6 (MPP6), which interacts with the synaptic scaffolding protein Lin7 and cell adhesion molecule 4 (CADM4) in Schwann cells that form myelin. In the present study, we investigated the localization of and proteins related to MPP2, a highly homologous family protein of MPP6, in the cerebellum of the mouse central nervous system, in which neurons are well organized. Immunostaining for MPP2 was observed at cerebellar glomeruli (CG) in the granular layer after postnatal day 14. Using the high-resolution Airyscan mode of a confocal laser-scanning microscope, MPP2 was detected as a dot pattern and colocalized with CADM1 and Lin7, recognized as small ring/line patterns, as well as with calcium/calmodulin-dependent serine protein kinase (CASK), NMDA glutamate receptor 1 (GluN1), and M-cadherin, recognized as dot patterns, indicating the localization of MPP2 in the excitatory postsynaptic region and adherens junctions of granule cells. An immunoprecipitation analysis revealed that MPP2 formed a molecular complex with CADM1, CASK, M-cadherin, and Lin7. Furthermore, the Lin7 staining pattern showed small rings surrounding mossy fibers in wild-type CG, while it changed to the dot/spot pattern inside small rings detected with CADM1 staining in MPP2-deficient CG. These results indicate that MPP2 influences the distribution of Lin7 to synaptic cell membranes at postsynaptic regions in granule cells at CG, at which electric signals enter the cerebellum. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved LIN-2 (CASK)/LIN-7 (Lin7A-C)/LIN-10 (APBA1) complex plays an important role in regulating spatial organization of membrane proteins and signaling components. In Caenorhabditis elegans, the complex is essential for the development of the vulva by promoting the localization of the sole Epidermal growth factor receptor (EGFR) ortholog LET-23 to the basolateral membrane of the vulva precursor cells where it can specify the vulval cell fate. To understand how the LIN-2/7/10 complex regulates receptor localization, we determined its expression and localization during vulva development. We found that LIN-7 colocalizes with LET-23 EGFR at the basolateral membrane, whereas the LIN-2/7/10 complex colocalizes with LET-23 EGFR at cytoplasmic punctae that mostly overlapwith the Golgi. Furthermore, LIN-10 recruits LIN-2, which in turn recruits LIN-7. We demonstrate that the complex forms in vivo with a particularly strong interaction and colocalization between LIN-2 and LIN-7, consistent with them forming a subcomplex. Thus, the LIN-2/7/10 complex forms on the Golgi on which it likely targets LET-23 EGFR trafficking to the basolateral membrane rather than functioning as a tether. [ABSTRACT FROM AUTHOR]

We here investigated the effects of overexpressed superoxide dismutase (SOD)1 and amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants G93A and G147S in Neuro 2A (N2A) cell lines, and found a three-fold increase in lamellipodia either in cells cultured under differentiated or undifferentiated growth conditions. In undifferentiated N2A cells, SOD1 constructs promoted lamellipodial protrusions to similar extent as the overexpression of Rac1, and SOD1-mediated lamellipodia were prevented by coexpression of the N17 dominant-negative form of Rac1, or shRNA for a downstream effector of Rac1, the insulin receptor tyrosine kinase substrate p53 (IRSp53) or its binding partner LIN7. Moreover, no additive effect was measured by coexpression of the SOD1 constructs with Rac1, IRSp53 or LIN7. Collectively these data support a role for SOD1 in the regulation of Rac1-mediated lamellipodia pathway, a property fully retained by the two SOD1 mutants.

The membrane skeletal complex, protein 4.1G–membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell–cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis. [ABSTRACT FROM AUTHOR]

We here investigated the effects of overexpressed superoxide dismutase (SOD)1 and amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants G93A and G147S in Neuro 2A (N2A) cell lines, and found a three-fold increase in lamellipodia either in cells cultured under differentiated or undifferentiated growth conditions. In undifferentiated N2A cells, SOD1 constructs promoted lamellipodial protrusions to similar extent as the overexpression of Rac1, and SOD1-mediated lamellipodia were prevented by coexpression of the N17 dominant-negative form of Rac1, or shRNA for a downstream effector of Rac1, the insulin receptor tyrosine kinase substrate p53 (IRSp53) or its binding partner LIN7. Moreover, no additive effect was measured by coexpression of the SOD1 constructs with Rac1, IRSp53 or LIN7. Collectively these data support a role for SOD1 in the regulation of Rac1-mediated lamellipodia pathway, a property fully retained by the two SOD1 mutants. [ABSTRACT FROM AUTHOR]

Plant acid invertases are considered to be the key enzymes in sucrose unloading and carbohydrate supply to sink tissues. Acid cell-wall invertases control sucrose transport via the apoplastic pathway during sink initiation and expansion. In this study, we identified 12 LIN7 gene homologs encoding cell-wall invertases in red- and green-fruited tomato accessions (Solanum section Lycopersicon) of self-compatible and self-incompatible species. All genes consisted of six exons and five introns, including highly conserved 9-bp exon II. Identification of 226 exonic single nucleotide polymorphisms as well as extremely high intron variability indicates a significant interspecific divergence among the examined tomato accessions. Computational prediction revealed protein structure typical for the glycosyl hydrolase family 32 and conserved catalytic sites described for other plant cell-wall invertases. LIN7 expression in mature buds and flowers confirms LIN7 role in the development of pollen tubes and grains. The variability in gene and protein sequences and species-specific differences in LIN7 expression patterns may be responsible for putative functional divergence of invertases. Furthermore, we performed phylogenetic analysis of the Solanum section Lycopersicon species based on the LIN7 gene, which clearly divided the analyzed tomato accessions into two main clusters corresponding to self-compatible and self-incompatible species and was in agreement with the separation into red- and green-fruited plants. Given that LIN7 plays an essential role in tomato fertility and fruit ripening, the characterization of protein variability within species of section Lycopersicon may be useful to evaluate the potential application of the encoding genes for tomato breeding programs. [ABSTRACT FROM AUTHOR]

Abstract: The vertebrate retina develops from an undifferentiated sheet of neuroepithelial cells, whose differentiation requires the generation and maintenance of the correct cellular polarity. To examine the role of cell polarity in retinal development, we cloned three zebrafish lin7 genes (lin7a, lin7b, and lin7c), which each encodes a protein candidate that is required for generation/maintenance of neuroepithelial cell junctions. These three zebrafish Lin7 proteins share over 78% amino acid identity and contain both L27 and PDZ domains that are present in all Lin7 homologs. Immunoblots revealed that the Lin7b and Lin7c proteins were first expressed in the developing eye by 24hr postfertilization (hpf), while Lin7a was not detected in the eye until 72 hpf. At 33 hpf, the Lin7 proteins localized at, or slightly apical of, the actin-associated adherens junctions in the retinal neuroepithelium. This subcellular distribution required the expression of the Nok protein. In the absence of Nok, the Lin7 proteins failed to localize to either the ectopic adherens junctions or the cell membrane. At 4 days postfertilization, in situ hybridisation revealed that all three lin7 genes were expressed in both the ganglion cell layer and the bipolar cell region of the inner nuclear layer. The lin7a gene was also expressed in the amacrine and horizontal cell regions of the inner nuclear layer, while lin7c was also expressed in the outer nuclear layer. In the adult retina, where Lin7a is the predominant form expressed, the Lin7 proteins were localized to the outer and inner plexiform layers, the bipolar and horizontal cells of the inner nuclear layer, and the ganglion cells. These results suggest that the three zebrafish Lin7 proteins possess partially redundant, yet essential, roles in retinal development. [Copyright &y& Elsevier]

Apicobasal polarity plays an important role in regulating asymmetric cell divisions by neural progenitor cells (NPCs) in invertebrates, but the role of polarity in mammalian NPCs is poorly understood. Here, we characterize the function of the PDZ domain protein MALS-3 in the developing cerebral cortex. We find that MALS-3 is localized to the apical domain of NPCs. Mice lacking all three MALS genes fail to localize the polarity proteins PATJ and PALS1 apically in NPCs, whereas the formation and maintenance of adherens junctions appears normal. In the absence of MALS proteins, early NPCs progressed more slowly through the cell cycle, and their daughter cells were more likely to exit the cell cycle and differentiate into neurons. Interestingly, these effects were transient; NPCs recovered normal cell cycle properties during late neurogenesis. Experiments in which MALS-3 was targeted to the entire membrane resulted in a breakdown of apicobasal polarity, loss of adherens junctions, and a slowing of the cell cycle. Our results suggest that MALS-3 plays a role in maintaining apicobasal polarity and is required for normal neurogenesis in the developing cortex. [ABSTRACT FROM AUTHOR]

Dendritic spines are the major locations of excitatory synapses in the mammalian central nervous system. The transformation from dendritic filopodia to dendritic spines has been recognized as one type of spinogenesis. For instance, syndecan-2 (SDC2), a synaptic heparan sulfate proteoglycan, is highly concentrated at dendritic spines and required for spinogenesis. It induces dendritic filopodia formation, followed by spine formation. However, the molecular regulation of the filopodium-spine transition induced by SDC2 is still unclear. In this report, we show that calcium is an important signal downstream of SDC2 in regulation of filopodium-spine transition but not filopodia formation. SDC2 interacted with the postsynaptic proteins calmodulin-dependent serine kinase (CASK) and LIN7 and further recruited NMDAR to the tips of filopodia induced by SDC2. Calcium influx via NMDAR promoted spine maturation because addition of EGTA or AP5 to the culture medium effectively prevented morphological change from dendritic filopodia to dendritic spines. Our data also indicated that F-actin rearrangement regulated by calcium influx is involved in the morphological change, because the knockdown of gelsolin, a calcium-activated F-actin severing molecule, impaired the filopodium-spine transition induced by SDC2. In conclusion, our study demonstrates that postsynaptic proteins coordinate to trigger calcium signalling and cytoskeleton rearrangement and consequently control filopodium-spine transition., (© 2014 Wiley Periodicals, Inc.)

Filopodia are dynamic, actin-rich finger-like structures that protrude from the cell membrane and play important roles in cell migration and neurite initiation and outgrowth. The insulin receptor substrate protein of 53 kDa (IRSp53) and the mammalian Diaphanous members of the formin family of proteins (mDia) are two key players in the formation of filopodia and neurites. IRSp53 is an adaptor protein that acts at the membrane:actin interface, coupling membrane deformation with F-actin polymerization. mDia formin proteins, instead, can nucleate and polymerize linear actin filaments. Emerging genetic and biochemical evidence indicate that there are multiple and independent pathways leading to filopodium and neurite formation, but the precise molecular components of these pathways remain ill-defined. We recently identified the PDZ domain-containing protein LIN7 as a novel regulator of IRSp53. We further showed that the association between these two proteins is required to promote the formation of filopodia and neurites independently from mDia formin proteins, highlighting novel mechanisms of filopodia and neurite formation.