Junlin Teng

Traumatic scratch injury in astrocytes triggers calcium influx to activate the JNK/c-Jun/AP-1 pathway and switch on GFAP expression.

Astrocyte activation is a hallmark of central nervous system injuries resulting in glial scar formation (astrogliosis). The activation of astrocytes involves metabolic and morphological changes with complex underlying mechanisms, which should be defined to provide targets for astrogliosis intervention. Astrogliosis is usually accompanied by an upregulation of glial fibrillary acidic protein (GFAP). Using an in vitro scratch injury model, we scratched primary cultures of cerebral cortical astrocytes and observed an influx of calcium in the form of waves spreading away from the wound through gap junctions. Using the calcium blocker BAPTA‐AM and the JNK inhibitor SP600125, we demonstrated that the calcium wave triggered the activation of JNK, which then phosphorylated the transcription factor c‐Jun to facilitate the binding of AP‐1 to the GFAP gene promoter to switch on GFAP upregulation. Blocking calcium mobilization with BAPTA‐AM in an in vivo stab wound model reduced GFAP expression and glial scar formation, showing that the calcium signal, and the subsequent regulation of downstream signaling molecules, plays an essential role in brain injury response. Our findings demonstrated that traumatic scratch injury to astrocytes triggered a calcium influx from the extracellular compartment and activated the JNK/c‐Jun/AP‐1 pathway to switch on GFAP expression, identifying a previously unreported signaling cascade that is important in astrogliosis and the physiological response following brain injury. GLIA 2013;61:2063–2077

14-3-3γ affects dynamics and integrity of glial filaments by binding to phosphorylated GFAP

Recent findings indicated a protective role of GFAP in ischemic brain, injured spinal cord, and in neurodegenerative disease. We previously demonstrated that 14-3-3γ, once thought to be neuronal specific, was up-regulated by ischemia in astrocytes and may play a specific protective role in astrocytes. Here we report that 14-3-3γ associates with both soluble and filamentous GFAP in a phosphorylation- and cell-cycle-dependent manner in primary cultured astrocytes. The amount of association increases during G2/M phase due to more phosphorylated GFAP. Moreover, this interaction is independent of vimentin, another type III intermediate filament protein in astrocytes which forms glial filaments with GFAP. A series of domain deletion mutants and substitution mutations at phosphorylation sites (from serine to alanine) on GFAP demonstrated that serine 8 in the head domain is essential for the direct association of GFAP to 14-3-3γ. Overexpression of 14-3-3γ destroyed the integrity and affected the movement of GFAP intermediate filaments. This data demonstrates that 14-3-3γ contributes to the regulation of dynamics of GFAP filaments, which may contribute to the stability of the cytoskeleton and the mechanisms of central nervous system neurodegenerative disease.

LRRC45 Is a Centrosome Linker Component Required for Centrosome Cohesion

During interphase, centrosomes are connected by a proteinaceous linker between the proximal ends of the centrioles, which is important for the centrosomes to function as a single microtubule-organizing center. However, the composition and regulation of centrosomal linker remain largely unknown. Here, we show that LRRC45 is a centrosome linker that localizes at the proximal ends of the centrioles and forms fiber-like structures between them. Depletion of LRRC45 results in centrosome splitting during interphase. Moreover, LRRC45 interacts with both C-Nap1 and rootletin and is phosphorylated by Nek2A at S661 during mitosis. After phosphorylation, both LRRC45 centrosomal localization and fiber-like structures are significantly reduced, which subsequently leads to centrosome separation. Thus, LRRC45 is a critical component of the proteinaceous linker between two centrioles and is required for centrosome cohesion.

Achieving λ/10 resolution CW STED nanoscopy with a Ti:Sapphire oscillator.

In this report, a Ti:Sapphire oscillator was utilized to realize synchronization-free stimulated emission depletion (STED) microscopy. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. With synchronization-free STED, we imaged 200 nm nanospheres as well as all three cytoskeletal elements (microtubules, intermediate filaments, and actin filaments), clearly demonstrating the resolving power of synchronization-free STED over conventional diffraction limited imaging. It also allowed us to discover that, Dylight 650, exhibits improved performance over ATTO647N, a fluorophore frequently used in STED. Furthermore, we applied synchronization-free STED to image fluorescently-labeled intracellular viral RNA granules, which otherwise cannot be differentiated by confocal microscopy. Thanks to the widely available Ti:Sapphire oscillators in multiphoton imaging system, this work suggests easier access to setup super-resolution microscope via the synchronization-free STED.

The Intracellular Transport and Secretion of Calumenin-1/2 in Living Cells

Calumenin isoforms 1 and 2 (calu-1/2), encoded by the CALU gene, belong to the CREC protein family. Calu-1/2 proteins are secreted into the extracellular space, but the secretory process and regulatory mechanism are largely unknown. Here, using a time-lapse imaging system, we visualized the intracellular transport and secretory process of calu-1/2-EGFP after their translocation into the ER lumen. Interestingly, we observed that an abundance of calu-1/2-EGFP accumulated in cellular processes before being released into the extracellular space, while only part of calu-1/2-EGFP proteins were secreted directly after attaching to the cell periphery. Moreover, we found the secretion of calu-1/2-EGFP required microtubule integrity, and that calu-1/2-EGFP-containing vesicles were transported by the motor proteins Kif5b and cytoplasmic dynein. Finally, we determined the export signal of calu-1/2-EGFP (amino acid positions 20–46) and provided evidence that the asparagine at site 131 was indispensable for calu-1/2-EGFP stabilization. Taken together, we provide a detailed picture of the intracellular transport of calu-1/2-EGFP, which facilitates our understanding of the secretory mechanism of calu-1/2.

SCG10 promotes non-amyloidogenic processing of amyloid precursor protein by facilitating its trafficking to the cell surface

The processing of amyloid precursor protein (APP) is a key event in the pathogenesis of Alzheimers disease. As certain cleavage pathways tend to occur in particular subcellular compartments, the processing of APP is greatly influenced by factors that regulate its trafficking. Here we report that SCG10 directly interacts with the KFFEQ motif of the APP intracellular domain and promotes the non-amyloidogenic processing of the APP. Knockdown of SCG10 led to decreases in α cleavage products, sAPPα and CTFα, while increases of both Aβ1-40 and Aβ1-42. Elevation of SCG10 induced APP accumulation in post-Golgi vesicles and on the cell surface by facilitating its secretory pathway. In addition, the APP processing was dependent on the palmitoylation-mediated membrane-anchoring of SCG10. Furthermore, elevation of SCG10 reduced Aβ accumulation and amyloid plaque formation in the hippocampus of APPswe/PS1dE9 mice. Taken together, these results show that SCG10 has a potential role in preventing and treating Alzheimers disease.

Cep57, a NEDD1-binding pericentriolar material component, is essential for spindle pole integrity

Formation of a bipolar spindle is indispensable for faithful chromosome segregation and cell division. Spindle integrity is largely dependent on the centrosome and the microtubule network. Centrosome protein Cep57 can bundle microtubules in mammalian cells. Its related protein (Cep57R) in Xenopus was characterized as a stabilization factor for microtubule-kinetochore attachment. Here we show that Cep57 is a pericentriolar material (PCM) component. Its interaction with NEDD1 is necessary for the centrosome localization of Cep57. Depletion of Cep57 leads to unaligned chromosomes and a multipolar spindle, which is induced by PCM fragmentation. In the absence of Cep57, centrosome microtubule array assembly activity is weakened, and the spindle length and microtubule density decrease. As a spindle microtubule-binding protein, Cep57 is also responsible for the proper organization of the spindle microtubule and localization of spindle pole focusing proteins. Collectively, these results suggest that Cep57, as a NEDD1-binding centrosome component, could function as a spindle pole- and microtubule-stabilizing factor for establishing robust spindle architecture.

Silkworm Coatomers and Their Role in Tube Expansion of Posterior Silkgland

Background Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation. Methodology/Principal Findings Here, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (α,β,β′, δ, ε, and ζ-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of α-, β′- and γ-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of α-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion. Conclusions/Significance The initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and would facilitate the establishment of silkworm PSG as an efficient bioreactor.

Hierarchical assembly of centriole subdistal appendages via centrosome binding proteins CCDC120 and CCDC68

In animal cells, the centrosome is the main microtubule-organizing centre where microtubules are nucleated and anchored. The centriole subdistal appendages (SDAs) are the key structures that anchor microtubules in interphase cells, but the composition and assembly mechanisms of SDAs are not well understood. Here, we reveal that centrosome-binding proteins, coiled-coil domain containing (CCDC) 120 and CCDC68 are two novel SDA components required for hierarchical SDA assembly in human cells. CCDC120 is anchored to SDAs by ODF2 and recruits CEP170 and Ninein to the centrosome through different coiled-coil domains at its N terminus. CCDC68 is a CEP170-interacting protein that competes with CCDC120 in recruiting CEP170 to SDAs. Furthermore, CCDC120 and CCDC68 are required for centrosome microtubule anchoring. Our findings elucidate the molecular basis for centriole SDA hierarchical assembly and microtubule anchoring in human interphase cells.

PACSIN1, a Tau-interacting Protein, Regulates Axonal Elongation and Branching by Facilitating Microtubule Instability

Background: The regulation of microtubule dynamics is crucial for forming and maintaining neuronal polarity. Results: PACSIN1 interacts with Tau in axons and reduces axonal elongation and branching by promoting microtubule dynamics. Conclusion: PACSIN1 acts as a Tau-binding partner in regulating microtubule dynamics and is required for axonal plasticity. Significance: Learning how PACSIN1 regulates microtubule dynamics is important for understanding its roles in neuronal development and nervous system disorders. Tau is a major member of the neuronal microtubule-associated proteins. It promotes tubulin assembly and stabilizes axonal microtubules. Previous studies have demonstrated that Tau forms cross-bridges between microtubules, with some particles located on cross-bridges, suggesting that some proteins interact with Tau and might be involved in regulating Tau-related microtubule dynamics. This study reports that PACSIN1 interacts with Tau in axon. PACSIN1 blockade results in impaired axonal elongation and a higher number of primary axonal branches in mouse dorsal root ganglia neurons, which is induced by increasing the binding ability of Tau to microtubules. In PACSIN1-blocked dorsal root ganglia neurons, a greater amount of Tau is inclined to accumulate in the central domain of growth cones, and it promotes the stability of the microtubule network. Taken together, these results suggest that PACSIN1 is an important Tau binding partner in regulating microtubule dynamics and forming axonal plasticity.