Number Of Microtubule-associated Proteins Research Articles

Non-cytopathic bovine viral diarrhoea virus 2 induces autophagy to enhance its replication.

Bovine viral diarrhoea virus (BVDV) is an important viral pathogen that has an economic impact on the livestock industry worldwide. Autophagy is one of the earliest cell-autonomous defence mechanisms against microbial invasion, and many types of viruses can induce autophagy by infecting host cells. The aim of this study was to identify the role of autophagy in the pathogenesis of non-cytopathic (ncp) BVDV2 infection. Madin-Darby bovine kidney (MDBK) cells were treated with ncp BVDV2, rapamycin, or 3-methyladenine (MA) and ncp BVDV2 and then incubated at 37°C for 24h. Cells were harvested, and the effects of autophagy were determined by transmission electron microscopy (TEM), confocal laser microscopy, western blotting and qRT-PCR. Apoptotic analysis was also performed using western blotting and flow cytometry. In ncp BVDV2-infected MDBK cells, more autophagosomes were observed by TEM, and the number of microtubule-associated protein 1 light chain 3B (LC3B) with green fluorescent protein puncta was also increased. The ncp BVDV2-infected cells showed significantly enhanced conversion of LC3-I to LC3-II, as well as upregulation of autophagy-related proteins, including ATG5 and Beclin 1, and substantial degradation of p62/SQSTM1. These results are similar to those induced by rapamycin, an autophagy inducer. E2 protein expression, which is associated with viral replication, increased over time in ncp BVDV2-infected cells. Inhibition of autophagy by 3-MA in ncp BVDV2-infected MDBK cells downregulated the expressions of LC3-II, ATG5 and Beclin 1 and prevented the degradation of p62/SQSTM1. Moreover, the expressions of phosphorylated Akt and procaspase-3 were significantly increased in ncp BVDV2-infected cells. In addition, the mRNA level of protein kinase R (PKR) was significantly reduced in ncp BVDV2-infected cells. Our results demonstrate that ncp BVDV2 infection induced autophagy in MDBK cells via anti-apoptosis and PKR suppression. Therefore, autophagy may play a role in establishing persistent infection caused by ncp BVDV.

Microtubule Bundles in the Adult Cardiomyocyte

The role of Microtubules (MTs) in mechanosignalling and mechanical properties has seen a recent surge of interest among the cardiac and skeletal muscle community. We recently reported that MTs buckle and bear load during the cardiac cycle. This has a significant effect on contractile mechanics, suggesting that myocyte function is tunable by any regulatory pathway that alters the mechanical behavior of MTs. Other work ongoing in our lab indicates that a number of Microtubule Associated Proteins (MAPs) are robustly upregulated in human heart disease. Multi-MT assemblies or bundles associated with these and other MAPs have been observed and studied extensively in other contexts, including unicellular motile structures and neuronal axons, but have not been examined in cardiac myocytes. The number of MTs involved, their geometry and the relative abundance of such structures compared to isolated MTs have significant implications for the mechanical properties of a myocyte. Using targeted overexpression of several MAPs in adult rat cardiomyocytes, we examine the structure, magnitude and prevalence of MT bundles by electron microscopy. We find that single MTs are the most prevalent MT elements in the unmanipulated myocyte, accompanied by comparatively rare and small groupings of 2-3 MTs with wide spacing. MTs are preferentially located at the interface between myofibril and mitochondria. Manipulation of MT binding partners can modestly increase the size and likelihood of MT bundles, and also dramatically reduces the inter-tubule spacing to be in-line with canonical bundle spacing. When we further characterized the functional consequences of MT bundling on cardiac contractility, we were surprised to find that the promotion of bundling, at least with certain MAPs, does not appear to impair contraction by reinforcing compliant microtubules, but rather uncouples the MTs from the process of contraction-associated buckling.

Recent Progress in Therapeutic Strategies for Ischemic Stroke.

Possible strategies for treating stroke include neuroprotection in the acute phase of cerebral ischemia and stem cell therapy in the chronic phase of cerebral ischemia. Previously, we have studied the temporal and spatial expression patterns of c-fos, hypoxia inducible factor-1α (HIF-1α), heat shock protein 70 (HSP70), and annexin V after 90 min of transient middle cerebral occlusion in rats and concluded that there is a time window for neuroprotection from 12 to 48 h after ischemia. In addition, we have estimated the neuroprotective effect of glial cell line-derived neurotrophic factor (GDNF) by injecting Sendai viral vector containing the GDNF gene into the postischemic brain. This Sendai virus-mediated gene transfer of GDNF showed a significant neuroprotective effect in the ischemic brain. Additionally, we have administered GDNF and hepatocyte growth factor (HGF) protein into the postischemic rat brain and estimated the infarct size and antiapoptotic and antiautophagic effects. GDNF and HGF significantly reduced infarct size, the number of microtubule-associated protein 1 light chain 3 (LC3)-positive cells, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick-end labeling (TUNEL)-positive cells, indicating that GDNF and HGF were greatly associated with not only the antiapoptotic effect but also the antiautophagic effects. Finally, we have previously transplanted undifferentiated iPSCs into the ipsilateral striatum and cortex at 24 h after cerebral ischemia. Histological analysis was performed at 14 and 28 days after cell transplantation, and we found that iPSCs could supply a great number of doublecortin-positive neuroblasts but also formed tridermal teratoma in the ischemic brain. Our results suggest that iPSCs have a potential to provide neural cells after ischemic brain injury if tumorigenesis is properly controlled. In the future, we will combine these strategies to develop more effective therapies for the treatment of strokes.

Autophagy enhancement contributes to the synergistic effect of vitamin D in temozolomide-based glioblastoma chemotherapy.

Temozolomide (TMZ), an alkylating agent, is recommended as the initial treatment for high-grade glioblastoma. TMZ is widely used, but its short half-life and the frequency of tumor resistance limit its therapeutic efficacy. In the present study, the anticancer effect of vitamin D (VD) combined with TMZ upon glioblastoma was determined, and the underlying mechanism of this effect was identified. Through cell viability, clonogenic and wound healing assays, the current study demonstrated that treatment of a C6 glioblastoma cell line with TMZ and VD resulted in significantly increased in vitro antitumor effects compared with either VD or TMZ alone. Autophagy, hypothesized to be the dominant mechanism underlying TMZ-based tumor cell death, was maximally activated in TMZ and VD co-treated C6 cells. This was demonstrated by ultrastructural observations of autophagosomes, increased size and number of microtubule-associated protein 1 light chain 3 (LC3) puncta and increased conversion of LC3-I to LC3-II. However, the extent of apoptosis was not significantly different between cells treated with TMZ and VD and those treated with TMZ alone. Addition of the autophagy inhibitor 3-methyladenine markedly inhibited the anticancer effect of TMZ and VD treatment, indicating that the chemosensitizing effect of VD in TMZ-based glioblastoma therapy is generated through enhancement of cytotoxic autophagy. TMZ and VD co-treatment also significantly inhibited tumor progression and prolonged survival duration in rat glioblastoma orthotopic xenograft models when compared with TMZ treatment alone. These in vivo results are concordant with the aforementioned in vitro results, together revealing that the combined use of TMZ and VD exerts synergistic antitumor effects on rat models of glioblastoma and may represent an effective therapeutic strategy.

Trans-Golgi protein p230/golgin-245 is involved in phagophore formation

p230/golgin-245 is a trans-Golgi coiled-coil protein that is known to participate in regulatory transport from the trans-Golgi network (TGN) to the cell surface. We investigated the role of p230 and its interacting protein, microtubule actin crosslinking protein 1 (MACF1), in amino acid starvation-induced membrane transport. p230 or MACF1 knock-down (KD) cells failed to increase the autophagic flow rate and the number of microtubule-associated protein 1 light chain 3 (LC3)-positive puncta under starvation conditions. Loss of p230 or MACF1 impaired mAtg9 recruitment to peripheral phagophores from the TGN, which was observed in the early step of autophagosome formation. Overexpression of the p230-binding domain of MACF1 resulted in the inhibition of mAtg9 trafficking in starvation conditions as in p230-KD or MACF1-KD cells. These results indicate that p230 and MACF1 cooperatively play an important role in the formation of phagophore through starvation-induced transport of mAtg9-containing membranes from the TGN. In addition, p230 itself was detected in autophagosomes/autolysosome with p62 or LC3 during autophagosome biogenesis. Thus, p230 is an important molecule in phagophore formation, although it remains unclear whether p230 has any role in late steps of autophagy.

Is the Cytoskeleton an Intracellular Receptor for Adrenomedullin and PAMP?

Classical transmembrane receptors have been described for both adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP). Through interactions with these membrane receptors, AM and PAMP exert a variety of endocrine, paracrine, and autocrine functions. In addition to these better known activities, recent publications have shown that both peptides can bind directly to the cytoskeleton resulting in important cellular physiological responses. In vitro and in vivo experiments show that the peptides bind to major components of the cytoskeleton: tubulin and kinesin for PAMP and a number of microtubule-associated proteins (MAPs) in the case of AM. Physiological experiments show that PAMP contributes to microtubule fluidity and increases kinesin speed. Lack of AM and PAMP results in hyperpolymerization of the cytoskeleton and a reduced motility of intracellular organelles. These data suggest that the cytoskeleton may have a novel function as an intracellular receptor, acting as the binding site and the signal transducer for specific peptide hormones such as PAMP.

Arabidopsis thaliana, a plant model organism for the neuronal microtubule cytoskeleton?

The microtubule cytoskeleton is an important component of both neuronal cells and plant cells. While there are large differences in the function of microtubules between the two groups of organisms, for example plants coordinate the ordered deposition of cellulose through the microtubule cytoskeleton, there are also some notable similarities. It is suggested that Arabidopsis thaliana, with its superior availability of knockout lines, may be a suitable model organism for some aspects of the neuronal microtubule cytoskeleton. Some cellular processes that involve the neuronal microtubule cytoskeleton including neurotransmitter signalling and neurotrophic support may have homologous processes in plant cells. A number of microtubule-associated proteins (MAPs) are conserved, including katanin, EB1, CLASP, spastin, gephyrin, CRIPT, Atlastin/RHD3, and ELP3. As a demonstration of the usefulness of a plant model system for neuronal biology, an analysis of plant tubulin-binding proteins was used to show that Charcot-Marie-Tooth disease type 2D and spinal muscular atrophy may be due to microtubule dysfunction and suggest that indeed the plant microtubule cytoskeleton may be particularly similar to that of motor neurons as both are heavily reliant upon motor proteins.

Nusap1 is essential for neural crest cell migration in zebrafish

Microtubules play important roles in mitotic spindle assembly and chromosome segregation to maintain normal cell cycle progression. A number of microtubule-associated proteins have been identified in epithelial and neural cell cultures; however, their physiological significance is not well characterized due to the lack of appropriate in vivo animal models. Nucleolar spindle-associated protein (NuSAP) is a microtubule-binding protein and is reported to be involved in mitosis by cell culture studies. In this report, we identified the zebrafish homologue of human NuSAP and investigated its expression profile and functions. Using in situ hybridization, we demonstrated that transcripts of zebrafish nusap1 are specifically expressed in the retina, forebrain, hindbrain and neural crest. When the in vivo expression of nusap1 was knocked down through antisense oligonucleotide morpholino technology, the morphants of nusap1 showed impaired morphogenesis in the trunk and yolk extension, implying the involvement of Nusap1 in cell migration. Mechanistic studies revealed that nusap1 morphants have an altered expression pattern of neural crest markers crestin and sox9b, but normal expression of blood vessel and notochord markers gata1 and shh. In addition, nusap1 mRNA injection caused serious apoptosis in retina and hindbrain tissue, and these phenotypes can be rescued by co-injection of morpholino against nusap1. These observations not only suggest a role for Nusap1 in connecting apoptosis with cell migration, but also provide strong evidences that Nusap1 is potentially involved in morphogenesis in vertebrates.

The Cytoskeleton in Oligodendrocytes

Oligodendrocytes have a complex cytoarchitecture and are characterized by an elaborate network of microtubules. They provide the tracks for organelle trafficking and the intracellular translocation of myelin-specific gene products. The integrity of the cytoskeleton is an essential determinant of the function and survival of oligodendrocytes. Microtubule growth and stability are regulated by microtubule-associated proteins. Oligodendrocytes contain a number of microtubule-associated proteins, including the tau proteins, which are developmentally regulated and especially prominent in the branching points of the cellular processes. Process outgrowth is regulated by the interaction of Fyn kinase with the cytoskeleton and by microtubule-severing proteins, such as stathmin. Alterations or disruption of the cytoskeleton and abundant abnormal aggregates of cytoskeletal proteins often accompany neurodegenerative diseases, and inclusion bodies, resembling protein aggregates found in neurons, are prominent in oligodendroglial lesions in white matter pathology. This review emphasizes the role of the cytoskeleton, particularly of microtubules and their associated proteins, in oligodendrocytes during developmental processes. Furthermore, recent data on protein aggregate formation in oligodendroglial cells, which might occur during aging and disease processes, are summarized.

Xnf7 Contributes to Spindle Integrity through Its Microtubule-Bundling Activity

Regulation of microtubule dynamics and organization in mitosis by a number of microtubule-associated proteins (MAPs) is required for proper bipolar spindle assembly, yet the precise mechanisms by which many MAPs function are poorly understood. One interesting class of MAPs is known to localize to the nucleus during interphase yet fulfill important spindle functions during mitosis. We have identified Xenopus nuclear factor 7 (Xnf7), a developmental regulator of dorsal-ventral patterning, as a microtubule-binding protein that also associates with the nuclear import receptor importin alpha/beta. Xnf7 localized to interphase nuclei and metaphase spindles both in Xenopus egg extracts and cultured cells. Xnf7-depleted spindles were hypersensitive to microtubule-depolymerizing agents. Functional characterization of Xnf7 revealed that it binds directly to microtubules, exhibits RING-finger-dependent E3-ubiquitin-ligase activity, and has C-terminal-dependent microtubule-bundling activity. The minimal microtubule-bundling domain of Xnf7 was sufficient to rescue the spindle-hypersensitivity phenotype. Thus, we have identified Xnf7 as a nuclear MAP whose microtubule-bundling activity, but not E3-ligase activity, contributes to microtubule organization and spindle integrity. Characterization of the multiple activities of Xnf7 may have implications for understanding human diseases caused by mutations in related proteins.

Alzheimer's disease: Microtubule-associated proteins 2 (MAP 2) are not components of paired helical filaments

In Alzheimer's disease, the most characteristic neuropathological changes are the formation of neurofibrillary tangles (NFT) and neuritic plaques (NP) characterized by the presence of bundles of paired helical filaments (PHF) that accumulate in the degenerating neurites and neuronal cell bodies. Although the protein composition of the PHF is ill-defined, a number of microtubule-associated proteins have been implicated in these lesions. Here we report results with an antiserum monospecific for the microtubule-associated protein MAP 2 which does not cross-react with any other microtubular protein. Immunostaining with this antibody of sections from an Alzheimer's brain show a strong reactivity with NFT but no reactivity at the level of the NP. On the other hand, immunostaining of Alzheimer's brain sections with another antibody specific for the microtubule-associated protein τ shows strong staining of PHF on both NFT and NP. These findings confirm the presence of the τ proteins in the PHF and strongly suggest that MAP 2 may not be a main structural component of the PHF. Labelling of NFT with the anti-MAP 2 antiserum suggests a non-specific binding of MAP 2 to the PHF during the process of NFT formation.