Intact Microtubules Research Articles

Taxol, a Microtubule Stabilizer, Improves Cardiac Functional Recovery during Postischemic Reperfusion in Rat in Vitro

Microtubule disruption contributes to cellular and organic dysfunction, and is implicated in ischemia/reperfusion (I/R) injury. The purpose of this study was to explore the effects of taxol, a microtubule stabilizer, on cardiac functional recovery during reperfusion. Left ventricular developed pressure, left ventricular end-diastolic pressure, maximal time derivatives of pressure and the severity of ventricular arrhythmias were analyzed in isolated rat heart. Microtubule structure was immunohistochemically measured. Apoptosis and necrosis was identified with TUNEL or TTC staining, respectively. Mitochondrial permeability transition pore (mPTP) mRNA expression was examined by real-time polymerase chain reactions. mPTP opening, reactive oxygen species (ROS), and oxidative enzyme activities were measured with fluorometric or spectrophotometric techniques. Intracellular calcium concentration ([Ca(2+) ](i) ) and Ca(2+) transients were examined by Fura-2-AM and Fluo-3-AM, respectively. Cytosolic cytochrome c, sarcoplasmic reticulum Ca(2+) -ATPase (SERCA2), ryanodine receptors (RyR), phospholamban (PLB), and PLB phosphorylation were analyzed by Western blot. Effective refractory period (ERP) and afterpotential-mediated activity were detected using microelectrode. Taxol improved the functional recovery of post-I/R. Taxol preserved the intact microtubule structure in reperfusion. mPTP mRNA expression was unchanged while the mPTP opening was reduced by taxol, and this effect was accompanied by the decreased ROS level caused by oxidative enzymes activities' changes. Taxol reduced apoptosis and the level of cytosolic cytochrome c in reperfusion. Taxol also promoted rapid recovery of [Ca(2+) ](i) , prevented reduction of the amplitude of Ca(2+) transients and shortened the decay time of Ca(2+) transients. The protein expression of SERCA2, RyR, and PLB remained unchanged in reperfusion. Taxol prevented the increase of Phospho-Thr17-PLB and Phospho-Ser16-PLB in reperfusion. In addition, taxol facilitated rapid recovery of ERP and counter-acted afterpotential-mediated activity. Taxol may effectively improve cardiac functional recovery during reperfusion via inhibiting mPTP opening, ameliorating abnormal calcium homeostasis, and reducing the substrates associated with arrhythmias.

Ligand oligomerization state controls Tie2 receptor trafficking and Angiopoietin-2 ligand-specific responses

Angiopoietin 1 (Ang1) is an activating ligand for the endothelial receptor tyrosine kinase Tie2, whereas Ang2 acts as a context-dependent agonist or antagonist that has a destabilizing effect on the vasculature. The molecular mechanisms responsible for the versatile functions of Ang2 are poorly understood. We show here that Ang2, but not Ang1, induces Tie2 translocation to the specific cell-matrix contact sites located at the distal end of focal adhesions. The Ang2-specific Tie2 translocation was associated with distinct Tie2 activation and downstream signals which differed from those of Ang1, and led to impaired cell motility and weak cell-matrix adhesion. We demonstrate that the different oligomeric or multimeric forms of the angiopoietins induce distinct patterns of Tie2 trafficking; the lower oligomerization state of native Ang2 was crucial for the Ang2-specific Tie2 redistribution, whereas multimeric structures of Ang1 and Ang2 induced similar responses. The Ang2-specific Tie2 trafficking to cell-matrix contacts was also dependent on the cell substratum, α2β1-integrin-containing cell-matrix adhesion sites and intact microtubules. Our data indicate that the different subcellular trafficking of Tie2-Ang2 and Tie2-Ang1 complexes generates ligand-specific responses in the angiopoietin-Tie signaling pathway, including modulation of cell-matrix interactions.

MAP1B Interaction with the FW Domain of the Autophagic Receptor Nbr1 Facilitates Its Association to the Microtubule Network

Selective autophagy is a process whereby specific targeted cargo proteins, aggregates, or organelles are sequestered into double-membrane-bound phagophores before fusion with the lysosome for protein degradation. It has been demonstrated that the microtubule network is important for the formation and movement of autophagosomes. Nbr1 is a selective cargo receptor that through its interaction with LC3 recruits ubiquitinated proteins for autophagic degradation. This study demonstrates an interaction between the evolutionarily conserved FW domain of Nbr1 with the microtubule-associated protein MAP1B. Upon autophagy induction, MAP1B localisation is focused into discrete vesicles with Nbr1. This colocalisation is dependent upon an intact microtubule network as depolymerisation by nocodazole treatment abolishes starvation-induced MAP1B recruitment to these vesicles. MAP1B is not recruited to autophagosomes for protein degradation as blockage of lysosomal acidification does not result in significant increased MAP1B protein levels. However, the protein levels of phosphorylated MAP1B are significantly increased upon blockage of autophagic degradation. This is the first evidence that links the ubiquitin receptor Nbr1, which shuttles ubiquitinated proteins to be degraded by autophagy, to the microtubule network.

The centriolar satellite protein Cep131 is important for genome stability.

The centrosome acts as a centre for microtubule organisation and plays crucial roles in cell polarity, migration, growth and division. Cep131 has recently been described as a basal body component essential for cilium formation, but its function in non-ciliogenic cells is unknown. We identified human Cep131 (also known as AZI1) in a screen for regulators of genome stability. We show that centrosomal localisation of Cep131 is cell-cycle-regulated and requires both an intact microtubule network and a functional dynein-dynactin transport system. Cep131 is recruited to centriolar satellites by PCM1, and localised to the centriolar core region by both pericentrin and Cep290. Depletion of Cep131 results in a reduction in proliferation rate, centriole amplification, an increased frequency of multipolar mitosis, chromosomal instability and an increase in post-mitotic DNA damage. These data therefore highlight the importance of human Cep131 for maintaining genomic integrity.

Characterization of the Microtubules Modified with Photochromic Molecules

Previously we have introduced photochromic molecules into the functional region of kinesin and succeeded to regulate the ATPase activity of kinesin reversibly by light irradiation. However, the regulation efficiency of the ATPase activity was not significant. It is well known that the contraction of skeletal muscle is regulated by calcium binding to the rail protein actin filament. The regulating system achieves high efficient switching. Therefore, it is expected that introducing artificial switching system into the rail protein microtubules enables high efficient regulation of kinesin motor activity. In this study, we tried to introduce photochromic molecules into the microtubules to regulate kinesin movement. First we modified the microtubules composed of wild type tubulin and measured gliding speed for kinesin using invitro motility assay under UV or VIS light irradiation, respectively. Gliding speeds of the microtubules modified with 4-phenylazophenyl maleimide (PAM) or 3,3-dimethyl-6’-nitro −1-[2- [3-(3- pyrroline −2,5-dion) −1-yl] propanoyloxy] ethylindoline −2-spiro-2’ (2H)- chromene (MASP) were almost identical to intact microtubules and also were not influenced by UV or VIS light irradiation. This result suggested that that these photochromic molecules incorporated into intrinsic reactive cysteine residues on the surface of microtubules did not affect kinesin motor activity. Subsequently, we prepared the 4 types of α-tubulin mutants (V406C, V410C, M414C, G417C), which have additional cysteine residues in the kinesin binding region. Microtubule concentration dependent kinesin ATPase activity for the mutant microtubules, V406C, V410C and M414 showed almost similar values to that of WT microtubules. On the other hand, ATPase activity for G417C mutant microtubule drastically decreased. The mutant microtubules were modified with photochromic molecules and the effect upon UV-VIS light irradiation for gliding speed and kinesin ATPase activity were studied.

Par6γ is at the mother centriole and controls centrosomal protein composition through a Par6α-dependent pathway

The centrosome contains two centrioles that differ in age, protein composition and function. This non-membrane bound organelle is known to regulate microtubule organization in dividing cells and ciliogenesis in quiescent cells. These specific roles depend on protein appendages at the older, or mother, centriole. In this study, we identified the polarity protein partitioning defective 6 homolog gamma (Par6γ) as a novel component of the mother centriole. This specific localization required the Par6γ C-terminus, but was independent of intact microtubules, the dynein/dynactin complex and the components of the PAR polarity complex. Par6γ depletion resulted in altered centrosomal protein composition, with the loss of a large number of proteins, including Par6α and p150(Glued), from the centrosome. As a consequence, there were defects in ciliogenesis, microtubule organization and centrosome reorientation during migration. Par6γ interacted with Par3 and aPKC, but these proteins were not required for the regulation of centrosomal protein composition. Par6γ also associated with Par6α, which controls protein recruitment to the centrosome through p150(Glued). Our study is the first to identify Par6γ as a component of the mother centriole and to report a role of a mother centriole protein in the regulation of centrosomal protein composition.

Opposing microtubule motors drive robust nuclear dynamics in developing muscle cells

Dynamic interactions with the cytoskeleton drive the movement and positioning of nuclei in many cell types. During muscle cell development, myoblasts fuse to form syncytial myofibers with nuclei positioned regularly along the length of the cell. Nuclear translocation in developing myotubes requires microtubules, but the mechanisms involved have not been elucidated. We find that as nuclei actively translocate through the cell, they rotate in three dimensions. The nuclear envelope, nucleoli and chromocenters within the nucleus rotate together as a unit. Both translocation and rotation require an intact microtubule cytoskeleton, which forms a dynamic bipolar network around nuclei. The plus- and minus-end-directed microtubule motor proteins, kinesin-1 and dynein, localize to the nuclear envelope in myotubes. Kinesin-1 localization is mediated at least in part by interaction with klarsicht/ANC-1/Syne homology (KASH) proteins. Depletion of kinesin-1 abolishes nuclear rotation and significantly inhibits nuclear translocation, resulting in the abnormal aggregation of nuclei at the midline of the myotube. Dynein depletion also inhibits nuclear dynamics, but to a lesser extent, leading to altered spacing between adjacent nuclei. Thus, oppositely directed motors acting from the surface of the nucleus drive nuclear motility in myotubes. The variable dynamics observed for individual nuclei within a single myotube are likely to result from the stochastic activity of competing motors interacting with a complex bipolar microtubule cytoskeleton that is also continuously remodeled as the nuclei move. The three-dimensional rotation of myotube nuclei may facilitate their motility through the complex and crowded cellular environment of the developing muscle cell, allowing for proper myonuclear positioning.

Caspase-8 isoform 6 promotes death effector filament formation independent of microtubules

Caspase-8 can trigger cell death following prodomain-mediated recruitment to the 'death-inducing signaling complex.' The prodomain consists of two death effector domain (DED) motifs that undergo homotypic interactions within the cell. Aside from mediating recruitment of procaspase-8, the prodomains have also been implicated in regulating cell survival, proliferation, death, senescence, differentiation, and substrate attachment. Here, we perform the initial characterization of a novel isoform of caspase-8, designated caspase-8 isoform 6 (Casp-8.6), which encodes both prodomain DEDs followed by a unique C-terminal tail. Casp-8.6 is detected in cells of the hematopoietic compartment as well as several other tissues. When Casp-8.6 expression is reconstituted in caspase-8-deficient cells, Casp-8.6 does not significantly impact cellular proliferation, contrasting with our previous results using a domain-defined 'DED-only' construct that lacks the C-terminal tail. Like the DED-only construct, Casp-8.6 also robustly forms 'death effector' filaments, but in contrast to the DED construct, it does not exhibit a dependence upon intact microtubules to scaffold filament formation. Both types of death effector filaments promote apoptosis when expressed in the presence of full length caspase-8 (isoform 1). Together, the results implicate Casp-8.6 as a new physiological modulator of apoptosis.

Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury

Due to their viscoelastic nature, white matter axons are susceptible to damage by high strain rates produced during traumatic brain injury (TBI). Indeed, diffuse axonal injury (DAI) is one of the most common features of TBI, characterized by the hallmark pathological profiles of axonal bulbs at disconnected terminal ends of axons and periodic swellings along axons, known as “varicosities.” Although transport interruption underlies axonal bulb formation, it is unclear how varicosities arise, with multiple sites accumulating transported materials along one axon. Recently, axonal microtubules have been found to physically break during dynamic stretch injury of cortical axons in vitro. Here, the same in vitro model was used in parallel with histopathological analyses of human brains acquired acutely following TBI to examine the potential role of mechanical microtubule damage in varicosity formation post-trauma. Transmission electron microscopy (TEM) following in vitro stretch injury revealed periodic breaks of individual microtubules along axons that regionally corresponded with undulations in axon morphology. However, typically less than a third of microtubules were broken in any region of an axon. Within hours, these sites of microtubule breaks evolved into periodic swellings. This suggests axonal transport may be halted along one broken microtubule, yet can proceed through the same region via other intact microtubules. Similar axonal undulations and varicosities were observed following TBI in humans, suggesting primary microtubule failure may also be a feature of DAI. These data indicate a novel mechanism of mechanical microtubule damage leading to partial transport interruption and varicosity formation in traumatic axonal injury.

Microtubule- and Dynein-Dependent Nuclear Trafficking of Rhesus Rhadinovirus in Rhesus Fibroblasts

We investigated the role of microtubules in rhesus rhadinovirus (RRV) nuclear trafficking in rhesus fibroblasts. Intact microtubules and microtubule dynamics are required for RRV trafficking to perinuclear regions. RRV trafficking was reduced by an inhibitor of the dynein motor and overexpression of dynamitin. Furthermore, RRV particles are colocalized with microtubules and dynein proteins. These results highlight the important roles of microtubules and dynein-dynactin complexes in the transport of RRV particles to nuclei during primary infection.

Agonists that Increase [Ca2+]i Halt the Movement of Acidic Cytoplasmic Vesicles in MDCK Cells

Translocation of vesicles within the cytoplasm is essential to normal cell function. The vesicles are typically transported along the microtubules to their destination. The aim of this study was to characterize the vesicular movement in resting and stimulated renal epithelial cells. MDCK cells loaded with either quinacrine or acridine orange, dyes taken up by acidic vesicles, were observed at 37°C in semiopen perfusion chambers. Time-lapse series were analyzed by Imaris software. Our data revealed vigorous movement of stained vesicles in resting MDCK cells. These movements seem to require intact microtubules because nocodazole leads to a considerable reduction of the vesicular movements. Interestingly, we found that extracellular ATP caused the vesicular movement to cease. This observation was obvious in time lapse. Similarly, other stimuli known to increase the intracellular Ca²⁺ concentration ([Ca²⁺](i)) in MDCK cells (increment in the fluid flow rate or arginine vasopressin) also reduced the vesicular movement. These findings were quantified by analysis of single vesicular movement patterns. In this way, ATP was found to reduce the lateral displacement of the total population of vesicles by 40%. Because all these perturbations increase [Ca²⁺](i), we speculated that this increase in [Ca²⁺](i) was responsible for the vesicle arrest. Therefore, we tested the effect of the Ca²⁺ ionophore, ionomycin (1 μM), which in the presence of extracellular Ca²⁺ resulted in a considerable and sustained reduction of vesicular movement amounting to a 58% decrease in average lateral vesicular displacement. Our data suggest that vesicles transported on microtubules are paused when subjected to high intracellular Ca²⁺ concentrations. This may provide an additional explanation for the cytotoxic effect of high [Ca²⁺](i).

Design and Synthesis of Resveratrol-Based Nitrovinylstilbenes as Antimitotic Agents

A new series of resveratrol analogues was designed, synthesized, and demonstrated to be tubulin polymerization inhibitors. Most of these compounds exhibited antiproliferative activity and inhibited in vitro tubulin polymerization effectively at concentrations of 4.4-68.1 and 17-62 μM, respectively. Flow cytometry studies showed that compounds 7c, 7e, and 7g arrested cells in the G2/M phase of the cell cycle. Immunocytochemistry revealed loss of intact microtubule structure in cells treated with 7c and 7e. Docking of compounds 7c and 7e with tubulin suggested that the A-ring of the compounds occupies the colchicine binding site of tubulin, which coordinates with Cys241, Leu242, Ala250, Val318, Val328, and I378, and that the nitrovinyl side chain forms two hydrogen bonds with the main loop of the β-chain at Asn249 and Ala250.

Role of SNX16 in the Dynamics of Tubulo-Cisternal Membrane Domains of Late Endosomes

In this paper, we report that the PX domain-containing protein SNX16, a member of the sorting nexin family, is associated with late endosome membranes. We find that SNX16 is selectively enriched on tubulo-cisternal elements of this membrane system, whose highly dynamic properties and formation depend on intact microtubules. By contrast, SNX16 was not found on vacuolar elements that typically contain LBPA, and thus presumably correspond to multivesicular endosomes. We conclude that SNX16, together with its partner phosphoinositide, define a highly dynamic subset of late endosomal membranes, supporting the notion that late endosomes are organized in distinct morphological and functional regions. Our data also indicate that SNX16 is involved in tubule formation and cholesterol transport as well as trafficking of the tetraspanin CD81, suggesting that the protein plays a role in the regulation of late endosome membrane dynamics.

Regulation of Purinergic Signaling in Biliary Epithelial Cells by Exocytosis of SLC17A9-dependent ATP-enriched Vesicles

ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 μm that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling.

Mitochondria Associate with P-bodies and Modulate MicroRNA-mediated RNA Interference

P-bodies are cytoplasmic granules that are linked to mRNA decay, mRNA storage, and RNA interference (RNAi). They are known to interact with stress granules in stressed cells, and with late endosomes. Here, we report that P-bodies also interact with mitochondria, as previously described for P-body-related granules in germ cells. The interaction is dynamic, as a large majority of P-bodies contacts mitochondria at least once within a 3-min interval, and for about 18 s. This association requires an intact microtubule network. The depletion of P-bodies does not seem to affect mitochondria, nor the mitochondrial activity to be required for their contacts with P-bodies. However, inactivation of mitochondria leads to a strong decrease of miRNA-mediated RNAi efficiency, and to a lesser extent of siRNA-mediated RNAi. The defect occurs during the assembly of active RISC and is associated with a specific delocalization of endogeneous Ago2 from P-bodies. Our study reveals the possible involvement of RNAi defect in pathologies involving mitochondrial deficiencies.

Role of Doublecortin and CAM Kinase-Like-1 (DCAMKL-1) in Hepatitis C Virus-Induced Hepatocarcinogenesis

Background:Hepatitis C virus (HCV) infection is a prominent risk factor for the development of hepatocellular carcinoma (HCC). Most solid tumors, including HCC, are believed to contain a subpopulation of poorly differentiated cancer stem-like cells (CSCs) that can initiate tumor formation and are relatively insensitive to antineoplastic chemotherapy. Because the putative intestinal and pancreatic stem cell marker DCAMKL-1 possesses microtubulepolymerizing activity and because intact microtubule filaments are required for efficient HCV replication, we hypothesize that DCAMKL-1 may be involved in HCV-induced hepatocarcinogenesis. Aim: Our aim is to delineate the role(s) of DCAMKL-1 in the development of HCV-induced hepatocellular carcinoma. Methods: We investigated our hypothesis using a selectable subgenomic replicon-expressing hepatoma cell line (GS5) and a corresponding control cell line (Huh7.5), immunofluorescence, immunohistochemistry, Western blot analysis, real-time RT-PCR, fluorescence-activated cell sorting (FACS) and a mouse xenograft model. Results: We demonstrate that HCV enhances expression of DCAMKL-1 and colocalizes with the replication complex attached microtubules. The siRNA-mediated inhibition of DCAMKL-1 expression concomitantly reduced HCV replication (In Vitro). The HCV+DCAMKL-1+ cells isolated from GS5 or DCAMKL-1+ cells from Huh7.5 formed spheroid bodies in a matrigel-based culture assay. Furthermore, we demonstrate that HCV induces retrodifferentiation of the replicon-expressing cells that results in much higher expression of α-fetoprotein and cytokeratin-19 in the tumor xenografts of GS5 cells compared to the control Huh7.5 tumors. Conclusion: DCAMKL-1 may represent a novel cellular target for the treatment of HCV infection and for directly targeting CSCs in HCC. We further demonstrate that HCV exhibits intrinsic ability to induce CSC features in liver-derived cells.

Role of cysteine residues in cell surface expression of the human riboflavin transporter-2 (hRFT2) in intestinal epithelial cells.

The water-soluble vitamin B2 (riboflavin, RF) is an essential micronutrient for normal cell function and survival. Recent studies have identified a role for the human riboflavin transporter-2 (hRFT2) in normal intestinal RF absorption. However, little is known about the cell biology of this transporter and specifically about the molecular determinant(s) that dictate its cell surface expression in human intestinal epithelial cells. Here we show that the full-length hRFT2 protein fused to green fluorescent protein (GFP) (GFP-hRFT2) is expressed exclusively at the apical membrane domain of Caco-2 cells. COOH-terminal sequence was essential in dictating cell surface expression with a specific role for conserved cysteine residues (C463 and C467). Mutation of C463 and C467 ablated RF uptake, explained by retention of the constructs within the endoplasmic reticulum. Modeling analysis suggested a potential disulfide bridge between C463 and C386. Consistent with this prediction, mutating the C386 site in the context of the full-length transporter resulted in intracellular retention, whereas mutation of another conserved cysteine (C326A) was without effect on hRFT2 targeting. Intracellular trafficking of hRFT2 was also examined and appeared to involve distinct vesicular structures, the motility of vesicles critically dependent on an intact microtubule network. These results demonstrate a potential role for specific cysteine residues in the cell surface expression of the hRFT2 in human intestinal epithelial cells.

Abstract 506: Mechanisms of microvesicle-mediated communication in cancer

Abstract The many functional roles of microvesicles (MVs) in tumor development and progression have lately become a subject of intense investigation. Cancer cell derived genetic material and signaling proteins residing in MVs can be transferred to recipient cells by as yet ill-defined mechanisms. We have found that heparan sulfate proteoglycans (HSPGs), which previously have been implicated as receptors of various macromolecular ligands, also can mediate the uptake of MVs. Our data suggest that MV uptake involves membrane raft, non-caveolin dependent endocytosis. These findings are consistent with recent data from our group on HSPG-mediated uptake of antibody-conjugated nanoparticles. Furthermore, MVs were found to be sorted to CD63-containing, vesicular compartments through intracellular transport that appeared highly dependent on an intact microtubule network. Intriguingly, preliminary findings support the concept of intracellular exchange of MV cargo, thus giving rise, in a single transporting vesicle, to a signal that integrates information from multiple cells. The presented results reveal a novel receptor that mediates MV uptake, and advance our general understanding of MV-dependent transfer of molecular information. Finally, the finding that MVs can potentially harbor molecules from multiple cells residing in the tumor environment opens up new, exciting roles of MVs as information carriers in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2011-506

Mechanism of Microtubule-facilitated “Fast Track” Nuclear Import

Although the microtubule (MT) cytoskeleton has been shown to facilitate nuclear import of specific cancer-regulatory proteins including p53, retinoblastoma protein, and parathyroid hormone-related protein (PTHrP), the MT association sequences (MTASs) responsible and the nature of the interplay between MT-dependent and conventional importin (IMP)-dependent nuclear translocation are unknown. Here we used site-directed mutagenesis, live cell imaging, and direct IMP and MT binding assays to map the MTAS of PTHrP for the first time, finding that it is within a short modular region (residues 82-108) that overlaps with the IMPβ1-recognized nuclear localization signal (residues 66-108) of PTHrP. Importantly, fluorescence recovery after photobleaching experiments indicated that disruption of the MT network or mutation of the MTAS of PTHrP decreases the rate of nuclear import by 2-fold. Moreover, MTAS functions depend on mutual exclusivity of binding of PTHrP to MTs and IMPβ1 such that, following MT-dependent trafficking toward the nucleus, perinuclear PTHrP can be displaced from MTs by IMPβ1 prior to import into the nucleus. This is the first molecular definition of an MTAS that facilitates protein nuclear import as well as the first delineation of the mechanism whereby cargo is transferred directly from the cytoskeleton to the cellular nuclear import apparatus. The results have broad significance with respect to fundamental processes regulating cell physiology/transformation.

The Mechanism for Subcellular Localization of Nuclear Receptor CAR

Animals including human beings have defense mechanisms against the toxicity of xenobiotics such as medicinal compounds and environmental pollutants. Receptor-type transcriptional factors, such as aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR), play important roles in the defense against xenobiotic toxicities. In the absence of stimuli, these receptors are distributed predominantly in the cytoplasmic compartment. Following xenobiotic stimuli, receptors translocate into the nucleus and transactivate its target genes. However, the exogenously expressed CAR translocates spontaneously into the nucleus in immortal cells. Previously, we identified subcellular localization signals in rat CAR: nuclear localization signal (NLS), nuclear export signal (NES) and cytoplasmic retention region (CRR). Lack of CRR function might be responsible for the spontaneous nuclear accumulation of CAR in immortal cells. Further, the nuclear import of CAR is regulated by the importin-Ran system, which is required for maintaining an intact microtubule network. Clarifying the mechanisms underlying the nuclear translocation of CAR would be useful for the establishment of novel assay systems for the screening of ligands and activators of CAR using immortal cells without sacrificing animals.