Microtubule Stability Research Articles

Impacts of vimentin on centrosome functioning and microtubule dynamics.

Cell migration is an important feature of migrating fibroblasts. For cell migration to occur, the cells must establish a polarity. The cytoskeletal filaments and the centrosome positioning play an important role in cell polarity. Intrinsically polarized microtubules are often associated with cell polarity. But recent studies show that loss of vimentin intermediate filaments (IF) leads to abnormally persistent cell migration in 3D microchannels. This result suggests vimentin also plays an important role in cell polarity. However, the interaction of vimentin IF with centrosome and microtubules to orchestrate cell polarity is unclear. To understand this, we investigate the effects of vimentin on the microtubule-nucleating activity of the cell centrosome and the dynamics of microtubule network using wild-type and vimentin-null mouse embryonic fibroblasts (mEFs). Our results indicate, the presence of vimentin impacts the structure of the centrosome by increasing the area of the centrosome marked by pericentriolar material (PCM) proteins. Vimentin also promotes microtubule regrowth after nocodazole washout assay and enhances the stability of microtubules by increasing the presence of acetylated (post translational modification) microtubules. Our findings give a new insight to the role of vimentin in cell polarity by modulating centrosome structure and functioning.

Characterization of pseudophosphorylated tau protein.

Tau protein facilitates assembly, organization, and stability of microtubules found in neuronal axons. Hyperphosphorylation results in a loss of tau function from decreased microtubule binding affinity which is prominent in Alzheimer's Disease. The current study employed 7-Phos—a tau mutant with seven specific site pseudophosphorylation mutations—to mimic the characteristic changes found in hyperphosphorylation. In vivo, tau has six naturally occurring isoforms that vary in the length of the N-terminal projection domains, three of which were investigated in this study. Biophysical characterization of these isoforms was accomplished with several techniques. SDS-PAGE was used to identify an upward band shift, a feature of 7-Phos. A thioflavin fluorescence assay was performed to compare the arachidonic acid induced polymerization of the 7-Phos variants with their wild-type counterparts. Atomic force microscopy was used to measure the length of 7-Phos tau polymers over several days and to analyze the intermolecular interactions between molecules of 7-Phos. We determined that the total amount of polymerization measured by thioflavin fluorescence of the two longer 7-Phos isoforms, 1N4R and 2N4R, were similar to their corresponding wild-type proteins. The total amount of polymerization of the shortest isoform, 0N4R, was greater than its wild-type. Atomic force microscopy (AFM) imaging revealed that 7-Phos isoforms form longer polymers than their wild-type. Atomic force microscopy of immobilized tau layers allowed comparison of projection domain lengths between 7-Phos and wild-type. These results demonstrate some novel AFM-based measurements to characterize tau polymerization, projection domain extension/conformation and intermolecular interactions.

Regional redistribution of CB1 cannabinoid receptors in human foetal brains with Down's syndrome and their functional modifications in Ts65Dn+/+ mice.

The endocannabinoid system with its type 1 cannabinoid receptor (CB1 R) expressed in postmitotic neuroblasts is a critical chemotropic guidance module with its actions cascading across neurogenic commitment, neuronal polarisation and synaptogenesis in vertebrates. Here, we present the systematic analysis of regional CB1 R expression in the developing human brain from gestational week 14 until birth. In parallel, we diagrammed differences in CB1 R development in Down syndrome foetuses and identified altered CB1 R signalling. Foetal brains with normal development or with Down's syndrome were analysed using standard immunohistochemistry, digitalised light microscopy and image analysis (NanoZoomer). CB1 R function was investigated by in vitro neuropharmacology from neonatal Ts65Dn transgenic mice brains carrying an additional copy of ~90 conserved protein-coding gene orthologues of the human chromosome 21. We detected a meshwork of fine-calibre, often varicose processes between the subventricular and intermediate zones of the cortical plate in the late first trimester, when telencephalic fibre tracts develop. The density of CB1 Rs gradually decreased during the second and third trimesters in the neocortex. In contrast, CB1 R density was maintained, or even increased, in the hippocampus. We found the onset of CB1 R expression being delayed by ≥1month in age-matched foetal brains with Down's syndrome. In vitro, CB1 R excitation induced excess microtubule stabilisation and, consequently, reduced neurite outgrowth. We suggest that neuroarchitectural impairments in Down's syndrome brains involve the delayed development and errant functions of the endocannabinoid system, with a particular impact on endocannabinoids modulating axonal wiring.

Characterizing tubulin interactions with the proline rich region of tau.

Tau is a microtubule-associated protein thought to function in the regulation of microtubule stability and dynamics. Normally, tau plays an important role in modulating axonal microtubules in neurons, where it is highly expressed. Intracellular tau aggregates are found in a broad class of disorders, including Alzheimer's Disease, termed tauopathies. As an intrinsically disordered protein, tau lacks stable secondary and tertiary structure, and this structural disorder is maintained even when binding to soluble tubulin and microtubules. Multiple tau-tubulin binding sites have also been identified, spanning the proline-rich region (PRR), microtubule binding repeats (MTBR: R1-R4), and pseudo-repeat, R′. Although dozens of post-translational modifications have been identified on tau, phosphorylation, and specifically hyperphosphorylation, of tau is correlated with disease and alterations to microtubule binding. Intriguingly, potential phosphorylation sites also cluster with high frequency within the PRR. Our lab has recently demonstrated that the isolated PRR both binds tubulin and has tubulin polymerization capacity. Here, we use fluorescence correlation spectroscopy (FCS) in conjunction with bulk polymerization measurements to characterize the impact of phosphomimic mutations in the PRR on tubulin binding and polymerization. We find that single phosphomimics affect tubulin binding nominally, with T231E having the most significant impact. However, as phosphorylation events rarely occur in isolation on tau, we also tested combinatorial effects. We found that multiple phosphomimics alter tubulin binding and microtubule polymerization. Furthermore, we used mass spectrometry to map the regions of the PRR involved in tubulin binding. Together these measurements provide insight into previously overlooked relevance of tau's PRR in functional interactions with tubulin.

Low-Dose Taxol Promotes Neuronal Axons Extension and Functional Recovery after Spinal Cord Injury.

Axonal regeneration has been the research focus in the field of clinical treatment for spinal cord injury (SCI). The growth and extension of neuronal axons is a dynamic biological process mediated by the cytoskeleton, and microtubule plays an important role in axonal growth. Moderate stabilization of microtubule promotes axonal growth and eliminates various intra- and extracellular mechanisms that impede axonal regeneration. After SCI, the damaged axons rapidly form a growth cone, wherein the stability of tubulin decreases, impairing axonal regeneration. Taxol with proven clinical safety is commonly used as a broad-spectrum antitumor drug. Importantly, Taxol can promote axonal extension by enhancing and stabilizing the microtubule assembly. In our study, we systematically investigated the differentiation of neural stem cells (NSCs) in vitro and functional recovery in injured rats in vivo following Taxol treatment. Low-dose Taxol promoted differentiation of NSCs to neurons and significantly extended the axons in vitro. In vivo, Taxol promoted the expression of βIII-tubulin in the injured areas and motor function recovery after SCI. Low-dose Taxol is a promising clinical agent to promote axonal regeneration after SCI.

Microtubule stabilization promotes the synthesis of type 2 collagen in nucleus pulposus cell by activating hippo-yap pathway.

Intervertebral disc degeneration (IDD) is the cardinal pathological mechanism that underlies low back pain. Mechanical stress of the intervertebral disc may result in a change in nucleus pulposus cells state, matrix degradation, and degeneration of the disc. Microtubules, which are components of the cytoskeleton, are involved in driving or regulating signal pathways, which sense and transmit mechano-transduction. Microtubule and the related proteins play an important role in the development of many diseases, while little is known about the role of microtubules in nucleus pulposus cells. Researchers have found that type II collagen (COL2) expression is promoted by microtubule stabilization in synovial mesenchymal stem cells. In this study, we demonstrated that microtubule stabilization promotes the expression of COL2 in nucleus pulposus cells. Stabilized microtubules stimulating Hippo signaling pathway, inhibiting YAP protein expression and activity. In addition, microtubules stabilization promotes the expression of COL2 and alleviates disc degeneration in rats. In summary, our study for the first time, identifies microtubule as a promising therapeutic target for IDD, up-regulating the synthesis of COL2 via Hippo-Yap pathway. Our findings may provide new insights into the etiologies and pathology for IDD, further, targeting of microtubule acetylation may be an effective strategy for the treatment of IDD.

Lissencephaly-1 mutations enhance traumatic brain injury outcomes in Drosophila.

Traumatic brain injury (TBI) outcomes vary greatly among individuals, but most of the variation remains unexplained. Using a Drosophila melanogaster TBI model and 178 genetically diverse lines from the Drosophila Genetic Reference Panel (DGRP), we investigated the role that genetic variation plays in determining TBI outcomes. Following injury at 20-27 days old, DGRP lines varied considerably in mortality within 24 h ("early mortality"). Additionally, the disparity in early mortality resulting from injury at 20-27 vs 0-7 days old differed among DGRP lines. These data support a polygenic basis for differences in TBI outcomes, where some gene variants elicit their effects by acting on aging-related processes. Our genome-wide association study of DGRP lines identified associations between single nucleotide polymorphisms in Lissencephaly-1 (Lis-1) and Patronin and early mortality following injury at 20-27 days old. Lis-1 regulates dynein, a microtubule motor required for retrograde transport of many cargoes, and Patronin protects microtubule minus ends against depolymerization. While Patronin mutants did not affect early mortality, Lis-1 compound heterozygotes (Lis-1x/Lis-1y) had increased early mortality following injury at 20-27 or 0-7 days old compared with Lis-1 heterozygotes (Lis-1x/+), and flies that survived 24 h after injury had increased neurodegeneration but an unaltered lifespan, indicating that Lis-1 affects TBI outcomes independently of effects on aging. These data suggest that Lis-1 activity is required in the brain to ameliorate TBI outcomes through effects on axonal transport, microtubule stability, and other microtubule proteins, such as tau, implicated in chronic traumatic encephalopathy, a TBI-associated neurodegenerative disease in humans.

In-depth genetic and molecular characterization of diaphanous related formin 2 (DIAPH2) and its role in the inner ear.

Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.

Tau phosphorylation and PAD exposure in regulation of axonal growth.

Introduction: Tau is a microtubule associated phosphoprotein found principally in neurons. Prevailing dogma continues to define microtubule stabilization as the major function of tau in vivo, despite several lines of evidence suggesting this is not the case. Most importantly, tau null mice have deficits in axonal outgrowth and neuronal migration while still possessing an extensive microtubule network. Instead, mounting evidence suggests that tau may have a major function in the regulation of fast axonal transport (FAT) through activation of neuronal signaling pathways. Previous studies identified a phosphatase activating domain (PAD) at the tau N-terminal that is normally sequestered, but is constitutively exposed in tauopathies. When exposed, the PAD activates a signaling cascade involving PP1 and GSK3β which affects cellular functions including release of cargo from kinesin. Furthermore, we discovered that PAD exposure can be regulated by a single phosphorylation at T205. Exposure of the PAD is an early event in multiple tauopathies and a major contributing factor to neurodegeneration associated with tau hyperphosphorylation. However, effects of tau PAD exposure on anterograde FAT raised the interesting possibility that this pathway may be a mechanism for physiological regulation of cargo delivery through site-specific phosphorylation of tau and transient activation of PP1 and GSK3β. Significantly, there is already evidence of local control of PP1 and GSK3β at sites which require cargo delivery. Methods: To investigate this hypothesis, first we evaluated cellular localization of tau PAD exposure, pT205 tau phosphorylation, and active GSK3β in primary hippocampal neurons during development. Second, we analyzed the axonal outgrowth of tau knockout neurons following transfection with full length hTau40-WT, hTau40-ΔPAD, or hTau40-T205A. Results and Discussion: The results presented here suggest that transient activation of a PP1-GSK3β signaling pathway through locally regulated PAD exposure is a mechanism for cargo delivery, and thereby important for neurite outgrowth of developing neurons.

Libertellenone T, a Novel Compound Isolated from Endolichenic Fungus, Induces G2/M Phase Arrest, Apoptosis, and Autophagy by Activating the ROS/JNK Pathway in Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third most deadly type of cancer in the world and continuous investigations are required to discover novel therapeutics for CRC. Induction of apoptosis is one of the promising strategies to inhibit cancers. Here, we have identified a novel compound, Libertellenone T (B), isolated from crude extracts of the endolichenic fungus from Pseudoplectania sp. (EL000327) and investigated the mechanism of action. CRC cells treated by B were subjected to apoptosis detection assays, immunofluorescence imaging, and molecular analyses such as immunoblotting and QRT-PCR. Our findings revealed that B induced CRC cell death via multiple mechanisms including G2/M phase arrest caused by microtubule stabilization and caspase-dependent apoptosis. Further studies revealed that B induced the generation of reactive oxygen species (ROS) attributed to activating the JNK signaling pathway by which apoptosis and autophagy was induced in Caco2 cells. Moreover, B exhibited good synergistic effects when combined with the well-known anticancer drug, 5-FU, and another cytotoxic novel compound D, which was isolated from the same crude extract of EL000327. Overall, Libertellenone T induces G2/M phase arrest, apoptosis, and autophagy via activating the ROS/JNK pathway in CRC. Thus, B may be a potential anticancer therapeutic against CRC that is suitable for clinical applications.

Morphological Evidence for Novel Roles of Microtubules in Macrophage Phagocytosis

Although the phagocytic activity of macrophages has long been studied, the involvement of microtubules in the process is not well understood. In this study, we improved the fixation protocol and revealed a dynamically rearranging microtubule network in macrophages, consisting of a basal meshwork, thick bundles at the cell edge, and astral microtubules. Some astral microtubules extended beneath the cell cortex and continued to form bundles at the cell edge. These microtubule assemblies were mutually exclusive of actin accumulation during membrane ruffling. Although the stabilization of microtubules with paclitaxel did not affect the resting stage of the macrophages, it reduced the phagocytic activity and membrane ruffling of macrophages activated with serum-MAF, which induced rapid phagocytosis. In contrast, the destabilization of microtubules with nocodazole enhanced membrane ruffling and the internalization of phagocytic targets suggesting an inhibitory effect of the microtubule network on the remodeling of the actin network. Meanwhile, the microtubule network was necessary for phagosome maturation. Our detailed analyses of cytoskeletal filaments suggest a phagocytosis control system involving Ca2+ influx, the destabilization of microtubules, and activation of actin network remodeling, followed by the translocation and acidification of phagosomes on the microtubule bundles.

CAMSAP2 localizes to the Golgi in islet β-cells and facilitates Golgi-ER trafficking

Glucose stimulation induces the remodeling of microtubules, which potentiates insulin secretion in pancreatic β-cells. CAMSAP2 binds to microtubule minus ends to stabilize microtubules in several cultured clonal cells. Here, we report that the knockdown of CAMSAP2 in primary β-cells reduces total insulin content and attenuates GSIS without affecting the releasability of insulin vesicles. Surprisingly, CAMSAP2 knockdown does not change microtubule stability. Unlike in cultured insulinoma cells, CAMSAP2 in primary β-cells predominantly localizes to the Golgi apparatus instead of microtubule minus ends. This novel localization is specific to primary β- but not α-cells and is independent of microtubule binding. Consistent with its specific localization at the Golgi, CAMSAP2 promotes efficientGolgi-ER trafficking in primary β-cells. Moreover, primary β-cells and insulinoma cells likely express different CAMSAP2 isoforms. We propose that a novel CAMSAP2 isoform in primary β-cells has a non-canonical function, which promotes Golgi-ER trafficking to support efficient production of insulin and secretion.

C. elegans XMAP215/ZYG-9 and TACC/TAC-1 act at multiple times during oocyte meiotic spindle assembly and promote both spindle pole coalescence and stability.

The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division. In C. elegans, XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibit multiple and indistinguishable oocyte spindle assembly defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect their requirements at high temporal resolution. Temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure, stabilizing pole foci as they grow and limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization that occurs upon nuclear envelope breakdown. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation. Third, we show that ZYG-9 and TAC-1 also are required for spindle assembly during meiosis II, independently of their meiosis I requirements. The metaphase pole stability requirement appears to be important for maintaining chromosome congression, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability.

Microtubule stabilization targeting regenerative chondrocyte cluster for cartilage regeneration.

Purpose: Chondrocytes (CHs) in cartilage undergo several detrimental events during the development of osteoarthritis (OA). However, the mechanism underlying CHs regeneration involved in pathogenesis is largely unknown. The aim of this study was to explore the underlying mechanism of regeneration of CHs involved in the pathological condition and the potential therapeutic strategies of cartilage repair. Methods and Materials: CHs were isolated from human cartilage in different OA stages and the high-resolution cellular architecture of human osteoarthritis was examined by applying single-cell RNA sequencing. The analysis of gene differential expression and gene set enrichment was utilized to reveal the relationship of cartilage regeneration and microtubule stabilization. Microtubule destabilizer (nocodazole) and microtubule stabilizer (docetaxel) treated-human primary CHs and rats cartilage defect model were used to investing the effects and downstream signaling pathway of microtubule stabilization on cartilage regeneration. Results: CHs subpopulations were identified on the basis of their gene markers and the data indicated an imbalance caused by an increase in the degeneration and disruption of CHs regeneration in OA samples. Interestingly, the CHs subpopulation namely CHI3L1+ CHs, was characterized by the cell regenerative capacity, stem cell potency and the activated microtubule (MT) process. Furthermore, the data indicated that MT stabilization was effective in promoting cartilage regeneration in rats with cartilage injury model by inhibiting YAP activity. Conclusion: These findings lead to a new understanding of CHs regeneration in the OA pathophysiology context and suggest that MT stabilization is a promising therapeutic target for OA and cartilage injury.

Colchicine attenuates the electrical remodeling of post-operative atrial fibrillation through inhibited expression of immune-related hub genes and stabilization of microtubules.

Rationale: Acute inflammation is a major risk factor for post-operative atrial fibrillation (POAF), and epicardial adipose tissue (EAT) is considered as a source of inflammatory mediators. However, underlying mechanisms and pharmacological targets of POAF are poorly understood. Methods: Integrative analysis of array data from EAT and right atrial appendage (RAA) samples was conducted to identify potential hub genes. Lipopolysaccharide (LPS)-stimulated inflammatory models in mice and in induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) were used to examine the exact mechanism underlying POAF. Electrophysiological analysis, multi-electrode array, and Ca2+ imaging was employed to explore the alterations of electrophysiology and Ca2+ homeostasis under inflammation. Flow cytometry analysis, histology and immunochemistry were performed to investigate immunological alterations. Results: We observed electrical remodeling, enhanced atrial fibrillation (AF) susceptibility, immune cell activation, inflammatory infiltration, and fibrosis in LPS-stimulated mice. LPS-stimulated iPSC-aCMs showed arrhythmias, abnormal Ca2+ signaling, reduced cell viability, disrupted microtubule network and increased α-tubulin degradation. VEGFA, EGFR, MMP9 and CCL2 were identified as hub genes simultaneously targeted in the EAT and RAA of POAF patients. Notably, treatment of colchicine in LPS-stimulated mice resulted in a U-shape dose-response curve, where greatly improved survival rates were observed only at doses between 0.10-0.40 mg/kg. At this therapeutic dose level, colchicine inhibited the expression of all the identified hub genes and effectively rescued the pathogenic phenotypes observed in LPS-stimulated mice and iPSC-aCM models. Conclusions: Acute inflammation promotes α-tubulin degradation, induces electrical remodeling, and both recruits and facilitates the infiltration of circulating myeloid cells. A certain dose of colchicine attenuates electrical remodeling and decreases the recurrence of AF.

Abnormal Hyperphosphorylation of Tau in Canine Immune-mediated Meningoencephalitis.

Tau is a microtubule-associated protein involved in the assembly and stabilization of microtubules. In human medicine, hyperphosphorylation of tau is associated with microtubule instability and is considered to play a role in the progression of multiple sclerosis (MS). MS is an autoimmune neurological disease that shares many characteristics, including pathological mechanisms, with canine meningoencephalitis of unknown etiology (MUE). With this background, this study investigated the presence of hyperphosphorylated tau in dogs with MUE and experimental autoimmune encephalomyelitis (EAE). In total, eight brain samples were examined from two neurologically normal dogs, three dogs with MUE, and three canine EAE models. Anti-(phospho-S396) tau antibody was used for immunohisto-chemistry, which stained hyperphosphorylated tau. In normal brain tissues, hyperphosphorylated tau was not found. In all the dogs with EAE and one of the dogs with MUE, immunoreactivity for S396 p-tau was observed in glial cell cytoplasm and the background in the periphery of the inflammatory lesion. These results suggest for the first time that tau pathology may be involved in the progression of neuroinflammation in dogs, similar to that in human MS.

Fusogenic liposome-coated nanoparticles for rapid internalization into donor corneal endothelial tissue to enable prophylaxis before transplantation.

Cold stress (hypothermia) during storage and cytokine stress due to acute allograft rejection adversely affect the donor corneal endothelium in the short term. Pharmacological pre-treatment (before transplantation) of the donor corneal endothelium or cells (propagated in vitro for cell injection therapy) with microtubule stabilizers, cold stress protectants, and other molecules is an attractive strategy to tackle damage caused by hypothermia and cytokine stress. These molecules can be delivered intracellularly to the donor corneal endothelium or cells at controlled rates for desired periods and with one-time administration using nanoparticles. However, the death-to-preservation time of donor corneas of more than 4 to 6 h significantly decreases endothelial cell density and increases the risk of microbial contamination. Therefore, we have developed fusogenic liposome-coated nanoparticles for rapid internalization of nanoparticles into cultured corneal endothelial cells and ex vivo corneal endothelial tissue. Here, we have shown that the fusogenic liposome-coated nanoparticles have the intrinsic ability to efficiently and rapidly internalize into cultured corneal endothelial cells and ex vivo corneal tissue within 3 h by possibly fusing with the cell membrane and bypassing the endocytic pathway. Lactate dehydrogenase assay showed that the internalized fusogenic liposome-coated nanoparticles did not cause cytotoxicity in endothelial cells associated with the ex vivo cornea for at least up to 2 days. Thus, fusogenic liposome-coated nanoparticles have great potential as a platform for engineering cells and endothelial tissue of donor corneas to facilitate prophylactic drug delivery during storage and after transplantation.

The interconnection of endoplasmic reticulum and microtubule and its implication in Hereditary Spastic Paraplegia

The endoplasmic reticulum (ER) and microtubule (MT) network form extensive contact with each other and their interconnection plays a pivotal role in ER maintenance and distribution as well as MT stability. The ER participates in a variety of biological processes including protein folding and processing, lipid biosynthesis, and Ca2+ storage. MTs specifically regulate cellular architecture, provide routes for transport of molecules or organelles, and mediate signaling events. The ER morphology and dynamics are regulated by a class of ER shaping proteins, which also provide the physical contact structure for linking of ER and MT. In addition to these ER-localized and MT-binding proteins, specific motor proteins and adaptor-linking proteins also mediate bidirectional communication between the two structures. In this review, we summarize the current understanding of the structure and function of ER-MT interconnection. We further highlight the morphologic factors which coordinate the ER-MT network and maintain the normal physiological function of neurons, with their defect causing neurodegenerative diseases such as Hereditary Spastic Paraplegia (HSP). These findings promote our understanding of the pathogenesis of HSP and provide important therapeutic targets for treatment of these diseases.

The function of ciliopathy protein FOP on cilia and cortical microtubule cytoskeleton in Euplotes amieti

FOP is a centriole satellite protein involved in ciliogenesis. Although centriole satellites are involved in centrosome and ciliumrelated protein trafficking, their functions related to ciliary assembly and maintenance of ciliary microtubule stability remain unclear. In this study, the function of the FOP gene in Euplotes amieti was investigated by interfering with its expression using RNAi. As a result, expression levels of the ciliary assembly-related proteins BBS8 and IFT88 were down-regulated. Swimming speeds also decreased and the Euplotes were only able to spin in circles, which suggested that the FOP protein is an important protein involved in ciliary motion. Further observations of Euplotes amieti microstructure and ultrastructure via immunofluorescence and transmission electron microscopy revealed that FOP not only participated in the formation of the ventral ciliary basal body but also played an important role in the maintenance of cortical microtubules, which is fundamental for the morphological structure of Euplotes amieti.

Oxidative stress induces tau hyperphosphorylation via MARK activation in neuroblastoma N1E-115 cells.

Reactive oxygen species are considered a cause of neuronal cell death in Alzheimer's disease (AD). Abnormal tau phosphorylation is a proven pathological hallmark of AD. Microtubule affinity-regulating kinases (MARKs) regulate tau-microtubule binding and play a crucial role in neuronal survival. In this study, we hypothesized that oxidative stress increases the phosphorylation of Ser262 of tau protein through activation of MARKs, which is the main reason for the development of AD. We investigated the relationship between tau hyperphosphorylation on Ser262 and MARKs in N1E-115 cells subjected to oxidative stress by exposure to a low concentration of hydrogen peroxide. This work builds on the observation that hyperphosphorylation of tau is significantly increased by oxidative stress. MARKs activation correlated with tau hyperphosphorylation at Ser262, a site that is essential to maintain microtubule stability and is the initial phosphorylation site in AD. These results indicated that MARKs inhibitors might serve a role as therapeutic tools for the treatment of AD.