Biomedical subjects

N Leclerc

Publications and source records attributed to N Leclerc.

At least 19 recordsLinked to original sources

PubMed2003

Altered levels and distribution of microtubule-associated proteins before disease onset in a mouse model of amyotrophic lateral sclerosis.

Alterations of the axonal transport and microtubule network are potential causes of motor neurodegeneration in mice expressing a mutant form of the superoxide dismutase 1 (SOD1G37R) linked to amyotrophic lateral sclerosis (ALS). In the present study, we investigated the biology of microtubule-associated proteins (MAPs), responsible for the formation and stabilization of microtubules, in SOD1G37R mice. Our results show that the protein levels of MAP2, MAP1A, tau 100 kDa and tau 68 kDa species decrease significantly as early as 5 months before onset of symptoms in the spinal cord of SOD1G37R mice, whereas decrease in levels of tau 52-55 kDa species is most often noted with the manifestation of the clinical symptoms. Interestingly, there was no change in the protein levels of MAPs in the brain of SOD1G37R mice, a CNS organ spared by the mutant SOD1 toxicity. Remarkably, as early as 5 months before disease onset, the binding affinities of MAP1A, MAP2 and tau isoforms to the cytoskeleton decreased in spinal cord of SOD1G37R mice. This change correlated with a hyperphosphorylation of the soluble tau 52-55 kDa species at epitopes recognized by the antibodies AT8 and PHF-1. Finally, a shift in the distribution of MAP2 from the cytosol to the membrane is detected in SOD1G37R mice at the same stage. Thus, alterations in the integrity of microtubules are early events of the neurodegenerative processes in SOD1G37R mice.

Actins↗

PubMed2002

Similar ultrastructural distribution of the 5-HT(2A) serotonin receptor and microtubule-associated protein MAP1A in cortical dendrites of adult rat.

As visualized by light and electron microscopic immunocytochemistry, the distribution of the neuronal serotonin-2A (5-HT(2A)) receptor is mainly intracellular throughout adult rat brain. This localization is particularly striking in the pyramidal cells of cerebral cortex, the dendrites of which are intensely immunoreactive, but without any labeling of their spines. In view of recent yeast two-hybrid and biochemical results suggesting an association of 5-HT(2A) receptors with the cytoskeletal microtubule-associated protein MAP1A, the respective subcellular distributions of the receptors and of MAP1A were compared by quantitative electron microscopic immunocytochemistry in dendrites of adult rat frontoparietal cortex. Counts of silver-intensified immunogold particles revealed a higher density of 5-HT(2A) receptors in smaller rather than larger dendrites, and an apportionment between pre-defined compartments representing the plasma membrane and the cytoplasm that was proportional to the relative surface area of these compartments. MAP1A immunoreactivity also predominated in smaller versus larger dendrites, but with a slightly lower proportion of labeling in the plasma membrane versus cytoplasmic compartment. The co-localization of 5-HT(2A) receptors and MAP1A protein in the same dendrites could be demonstrated in double immunolabeling experiments. These results confirmed the predominantly somato-dendritic, intracellular localization of 5-HT(2A) receptors in cerebral cortex, showed their higher concentration in distal as opposed to proximal dendrites, and suggested their potential association to the cytoskeleton in cortical neurons in vivo. Such a distribution of 5-HT(2A) receptors reinforces our earlier hypothesis that 5-HT(2A) receptors participate in intraneuronal signaling processes involving the cytoskeleton, and raises the possibility that their activation could be dependent upon that of another co-localized, plasma membrane-bound, 5-HT receptor.

Animals↗

PubMed2001

Selective changes in mitochondria respiratory properties in oxidative or glycolytic muscle fibers isolated from G93AhumanSOD1 transgenic mice.

Cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in cytosolic copper, zinc superoxide dismutase (SOD1). Total SOD activity and functional mitochondrial properties were studied in muscles and nervous tissues of control and transgenic mice mimicking the disease. It was found that total SOD activity was lower in nervous tissues than in muscles in both transgenic and control mice. In addition SOD activity increased during progression of disease in muscle but not in nervous tissue of transgenic mice. Maximal oxygen consumption and apparent Km for ADP were decreased in mitochondria from transgenic soleus (an oxidative muscle). However there was no difference between control and transgenic mice in respiratory parameters of mitochondria in the EDL muscle (a glycolytic muscle). These findings indicate that oxidative stress due to SOD1 mutations could alter energy metabolism in FALS mice, thereby affecting primarily oxidative muscle of the limbs, independently of motoneuron loss.

Adenosine Diphosphate↗

PubMed2000

Exogenous BDNF, NT-3 and NT-4 differentially regulate neurite outgrowth in cultured hippocampal neurons.

Multiple growth factors contribute to the differentiation of dendritic and axonal processes by a neuron. Cultured hippocampal cells elaborate dendritic and axonal processes following well-defined steps. We used this culture system to determine the specific effects of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) on dendritic and axonal differentiation in hippocampal pyramidal neurons. We demonstrated that each of these neurotrophins exert distinct effects on neurite outgrowth. Both BDNF and NT-3 had positive effects on the outgrowth of undifferentiated neurites, called minor neurites, and on the axonal process of hippocampal pyramidal neurons. However, the effect of NT-3 was more important than that of BDNF. On the other hand, NT-4 did not enhance axonal outgrowth but had only an effect on the outgrowth of minor neurites. Since cytoskeletal proteins play crucial roles in promoting neurite outgrowth, we examined the protein levels of some of these proteins that are associated with neurite outgrowth: beta-actin, gamma-actin, alpha-tubulin, MAP2 and tau. Surprisingly, we did not detect any change in their protein levels. Taken together, our results show that BDNF, NT-3 and NT-4 exert distinct effects on the neuritic compartments of hippocampal neurons.

Animals↗

PubMed2000

Expression of the AMF/neuroleukin receptor in developing and adult brain cerebellum.

The peptide sequence of autocrine motility factor (AMF), a tumor secreted cytokine that induces cell motility, corresponds to that of the previously identified cytokine/enzyme, neuroleukin/glucose-6-phosphate isomerase. Neuroleukin is a neurotrophic factor that promotes neuronal survival and sprouting at the neuromuscular junction. The AMF receptor (AMF-R) has been identified and shown to be highly expressed in malignant tumors with minimal expression in adjacent normal tissue. Neuroleukin mRNA is highly expressed in the cerebellum and we therefore undertook a developmental study of AMF-R expression in rat cerebellum. As determined by immunoblot, AMF-R is expressed at equivalent high levels in brain and cerebellum of postnatal day 5 (P5) and 12 (P12) rats and at significantly reduced levels in the adult. Coimmunofluorescence studies with MAP-2 and gamma-actin revealed that at P12, AMF-R was mainly localized to Purkinje and granule cells. Moreover, the premigratory cells of the external granular layer were also immunoreactive for AMF-R suggesting a role for AMF-R in granule cell migration during cerebellar development in the first two weeks after birth. In the adult, AMF-R distribution was similar to P12, although weaker, and was localized to Purkinje and granule cells. AMF-R labeling of GFAP positive glial processes could not be detected in cerebellar sections although in cerebellar primary cultures, both neurons and glial cells were labeled for AMF-R. In neurons, AMF-R labeling was present in the cell body, neurites and growth cones. These data indicate that regulation of the neurotrophic function of neuroleukin might be regulated spatially and temporally by expression of its receptor, AMF-R, in developing and adult cerebellum.

Actins↗

PubMed2000

Prevention of mutant SOD1 motoneuron degeneration by copper chelators in vitro.

An animal model of familial amyotrophic lateral sclerosis (FALS) has been generated by overexpression of human CuZn superoxide dismutase (SOD1) containing a substitution of glycine to alanine at position 93 in transgenic G93A mice. The loss of motoneurons shown in this model has been attributed to a dominant gain of function of this mutated enzyme, which might be due to copper toxicity. This hypothesis was tested in purified spinal motoneurons cultures originating from G93A transgenic embryos. Spinal motoneurons were isolated from E13 embryos by several steps including density gradient centrifugation. The effect of copper chelators on survival and neurite growth of motoneurons was investigated. Survival of G93A motoneurons was decreased by 46% as compared to wild-type motoneurons. Moreover, G93A motoneurons showed reduced neurite outgrowth. Copper chelators strikingly increased viability of G93A motoneurons (by over 200%) but had no effect on wild-type cells. Presence of DDC in the medium increases the length of neurites from G93A motoneurons. The present results suggest the capacity of copper chelators to reduce the effect of reverse function of mutated SOD1 on motoneurons.

Amyotrophic Lateral Sclerosis↗

PubMed1999

Inactivation of Rho signaling pathway promotes CNS axon regeneration.

Regeneration in the CNS is blocked by many different growth inhibitory proteins. To foster regeneration, we have investigated a strategy to block the neuronal response to growth inhibitory signals. Here, we report that injured axons regrow directly on complex inhibitory substrates when Rho GTPase is inactivated. Treatment of PC12 cells with C3 enzyme to inactivate Rho and transfection with dominant negative Rho allowed neurite growth on inhibitory substrates. Primary retinal neurons treated with C3 extended neurites on myelin-associated glycoprotein and myelin substrates. To explore regeneration in vivo, we crushed optic nerves of adult rat. After C3 treatment, numerous cut axons traversed the lesion to regrow in the distal white matter of the optic nerve. These results indicate that targeting signaling mechanisms converging to Rho stimulates axon regeneration on inhibitory CNS substrates.

ADP Ribose Transferases↗

PubMed1999

Dystonin-deficient mice exhibit an intrinsic muscle weakness and an instability of skeletal muscle cytoarchitecture.

Dystonia musculorum (dt) was originally described as a hereditary sensory neurodegeneration syndrome of the mouse. The gene defective in dt encodes a cytoskeletal linker protein, dystonin, that is essential for maintaining neuronal cytoskeletal integrity. In addition to the nervous system, dystonin is expressed in a variety of other tissues, including muscle. We now show that dystonin cross-links actin and desmin filaments and that its levels are increased during myogenesis, coinciding with the progressive reorganization of the intermediate filament network. A disorganization of cytoarchitecture in skeletal muscle from dt/dt mice was observed in ultrastructural studies. Myoblasts from dt/dt mice fused to form myotubes in culture; however, terminally differentiated myotubes contained incompletely assembled myofibrils. Another feature observed in dt/dt myotubes in culture and in skeletal muscle in situ was an accumulation and abnormal distribution of mitochondria. The diaphragm muscle from dt/dt mice was weak in isometric contractility measurements in vitro and was susceptible to contraction-induced sarcolemmal damage. Altogether, our data indicate that dystonin is a cross-linker of actin and desmin filaments in muscle and that it is essential for establishing and maintaining proper cytoarchitecture in mature muscle.

Actins↗

PubMed1999

Protection of immunoreactivity of dry immobilized proteins on microtitration plates in ELISA: application for detection of autoantibodies in myasthenia gravis.

We show the ability of the BSA-trehalose film to convert normally fragile proteins such as mouse monoclonal antibody to the Alzheimer precursor protein A4 (APP695) and cell line TE671 acetylcholine receptor (AChRTE671) into a stable reagent, after its immobilization on microtitration plates. The remarkable property of the dry immobilized proteins are their stability under prolonged exposure to temperatures as high as 50 degrees C. Using the AChRTE671, the proposed method was applied for the measurement of anti-AChR autoantibodies in Myasthenia gravis by means of an enzyme-linked immunosorbent assay (ELISA). The test was shown to be specific and able to detect anti-AChR autoantibodies at concentrations as low as 3 nM. Using the same AchRTE671 as antigen, the results of examination of 34 serum samples for detection of anti-AChR autoantibodies by ELISA were compared with those of the conventional radioimmunoprecipitation assay (RIA). It was concluded that ELISA is another useful method for the diagnosis of M. gravis. The ELISA method offers a rapid, simple, safe and inexpensive means for mass screening of M. gravis.

Autoantibodies↗

PubMed1999

Taxol selectively blocks microtubule dependent NF-kappaB activation by phorbol ester via inhibition of IkappaBalpha phosphorylation and degradation.

Activation of the NF-kappa-B transcription factors has been shown to be directly influenced by changes in the microtubule cytoskeleton network. To better understand cytoskeletal regulation of NF-kappaB, experiments were performed to determine whether the microtubule (MT) stabilizing agent taxol could modulate NF-kappaB activation in the presence of different NF-kappa-B inducers. Pretreatment of murine NIH3T3 and human 293 cells with 5 microM taxol resulted in complete inhibition of phorbol, 12-myristate, 13-acetate (PMA) mediated NF-kappaB activation, detected as the loss of DNA binding and reduced NF-kappaB dependent reporter gene activity. Furthermore, in COS-7 and NIH3T3 cells, PMA-induced Ikappa-Balpha turnover was dramatically reduced in taxol treated cells, mediated via the inhibition of IkappaBalpha phosphorylation. However, taxol did not prevent TNF-alpha induced Ikappa-Balpha phosphorylation, degradation, or NF-kappaB activation, indicating that TNF-alpha acts through a microtubule-independent pathway. In vitro kinase assays with PMA stimulated cell extracts demonstrated that taxol reduced protein kinase C activity by 30%, thus implicating the loss of PKC activity as a possible regulatory target of taxol-mediated suppression of NF-kappa-B. Since PMA causes modulation of cytoarchitecture through PKC activation, microtubule integrity and cell morphology was analysed by indirect immunofluorescence. Both PMA and nocodazole, a MT depolymerizing agent, caused microtubule depolymerization, whereas TNF-alpha did not alter MT integrity; concomitant taxol treatment blocked both nocodazole and PMA induced depolymerization of MTs, as well as NF-kappaB induction, thus demonstrating a link between microtubule depolymerization and NF-kappaB activation. These observations illustrate a novel biological activity of taxol as a selective inhibitor of NF-kappa-B activity, suggesting a link between the state of microtubule integrity and gene regulation.

3T3 Cells↗

PubMed1999

Tau-mediated process outgrowth is differentially altered by the expression of MAP2b and MAP2c in Sf9 cells.

It is well documented that the MAPs, MAP2 and tau, play pivotal roles in neurite outgrowth. Several isoforms of MAP2 and tau are coexpressed in neurons, suggesting that the pattern of neurite outgrowth results from a functional equilibrium among these isoforms. In the present study, by coexpressing two of these MAPs at the same time in Sf9 cells, we demonstrated that tau-mediated process outgrowth is affected differently by MAP2b and MAP2c. MAP2b impairs tau ability to induce process outgrowth. Tau affects MAP2c capacity to induce the formation of multiple processes. There is evidence that actin microfilaments (F-actin) are involved in the elaboration of tau-mediated process outgrowth in Sf9 cells. We compared the effects of MAP2b and MAP2c with the effects of tau on F-actin distribution and stability in Sf9 cells. In MAP2b- and MAP2c-expressing cells with processes, F-actin was redistributed. However, in MAP2b-expressing cells without processes, the distribution of F-actin appears to be similar to the one in wild-type infected cells. Collectively, these results indicate that MAP2b could impair the ability of MAP2c and tau to redistribute F-actin in Sf9 cells, thereby decreasing their capacity to induce process formation. Furthermore, MAP2b and MAP2c patterns of process outgrowth were differentially modified by depolymerization of F-actin by cytochalasin D (CD). As previously reported for tau-expressing cells, the MAP2b-expressing cells developed a higher number of processes per cell and a higher number of cells presented processes in the presence of CD. However, the number of cells with multiple processes was lower in MAP2b-expressing cells than in tau-expressing cells treated with CD at 24 h postinfection. This suggests that MAP2b exerts an effect on F-actin stability at an earlier stage of infection than tau. MAP2c had also some stabilizing effects on F-actin at an early stage of infection, since the percentage of cells presenting one process was similar to the nontreated cells. Therefore, MAP2b seems to have less capacity than MAP2c to redistribute F-actin but, nonetheless, both of these MAP2 isoforms exert a stabilizing effect on F-actin at an early stage of infection. Finally, by modifying phosphorylation we showed that MAP2c capacity to induce multiple processes is related to protein phosphorylation in Sf9 cells. Therefore, the differential effect of MAP2c and MAP2b on process outgrowth seems also to depend on protein phosphorylation.

Actins↗

PubMed1998

Increased beta-actin expression in an invasive moloney sarcoma virus-transformed MDCK cell variant concentrates to the tips of multiple pseudopodia.

An invasive variant of Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK-INV), which was isolated by the repeated selection of cells that successfully traversed a Matrigel-coated filter, exhibits increased motile ability and presents an elongated cell shape and numerous pseudopodia. Although stress fibers are present in both MDCK and MSV-MDCK cells, MSV-MDCK-INV cells contain no stress fibers and exhibit a dense concentration of actin at the tips of pseudopodia. Relative to both MDCK and MSV-MDCK cells, the MSV-MDCK-INV cells exhibit increased expression of beta-actin and redistribution of beta-actin to the tips of pseudopodia. These actin concentrations are enriched in both F- and G-actin and, thus, represent dynamic regions of actin cytoskeleton remodeling. The acquisition of invasive properties by epithelial transformants is, therefore, associated with the increased expression of beta-actin and its concentration in actin-rich domains, which may drive pseudopodial extension and facilitate tumor cell invasion.

Actins↗

PubMed1998

Dystonin is essential for maintaining neuronal cytoskeleton organization.

The mouse neurological mutant dystonia musculorum (dt) suffers from a hereditary sensory neuropathy. We have previously described the cloning and characterization of the dt gene, which we named dystonin (Dst). We had shown that dystonin is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin-binding domain. It has been shown previously that dystonin is a cytoskeletal linker protein, forming a bridge between F-actin and intermediate filaments. Here, we have used two different antibody preparations against dystonin and detected a high-molecular-weight protein in immunoblot analysis of spinal cord extracts. We also show that this high-molecular-weight protein was not detectable in the nervous system of all dt alleles tested. Immunohistochemical analysis revealed that dystonin was present in different compartments of neurons--cell bodies, dendrites, and axons, regions which are rich in the three elements of the cytoskeleton (F-actin, neurofilaments, and microtubules). Ultrastructural analysis of dt dorsal root axons revealed disorganization of the neurofilament network and surprisingly also of the microtubule network. In this context it is of interest that we observed altered levels of the microtubule-associated proteins MAP2 and tau in spinal cord neurons of different dt alleles. Finally, dt dorsal root ganglion neurons formed neurites in culture, but the cytoskeleton was disorganized within these neurites. Our results demonstrate that dystonin is essential for maintaining neuronal cytoskeleton integrity but is not required for establishing neuronal morphology.

Animals↗

PubMed1998

beta-Actin is confined to structures having high capacity of remodelling in developing and adult rat cerebellum.

Neurons undergo complex morphological changes during differentiation and in cases of plasticity. A major determinant of cell morphology is the actin cytoskeleton, which in neurons is comprised of two actin isoforms, non-muscle gamma- and beta-actin. To better understand their respective roles during differentiation and plasticity, their cellular and subcellular localization was examined in developing and adult cerebellar cortex. It was observed that gamma-actin is expressed at a constant level throughout development, while the level of beta-actin expression rapidly decreases with age. At the light microscopic level, gamma-actin staining is ubiquitous and the only developmental change observed is a relative reduction of its concentration in cell bodies and white matter. In contrast, beta-actin staining almost completely disappears from the cytoplasm of cell bodies, primary dendrites and axons. In young cerebellar cultures, gamma-actin is found in the cell body, neurites and growth cones, while beta-actin is mainly found in growth cones, as previously reported in other primary neuronal culture systems [Kaech et al. (1997), J. Neuroscience, 17, 9565-9572; Bassell et al., (1998), J. Neuroscience, 18, 251-265]. Electron microscopy of post-embedding immunogold-labelled tissue confirms the widespread distribution of gamma-actin, and also reveals an increased concentration of gamma-actin in dendritic spines in the adult. During development, beta-actin accumulation is observed in actively growing structures, e.g., growth cones, filopodia, cell bodies and axonal tracts. In the adult cerebellar cortex, beta-actin is preferentially found in dendritic spines, structures which are known to retain their capacity for morphological modifications in the adult brain. This differential subcellular localization and developmental regulation of the two actin isoforms point to their different roles in neurons.

Actins↗

PubMed1998

Time-gated autofluorescence microscopy of motile green microalga in an optical trap.

Ultrafast time-gated fluorescence imaging of optically trapped single motile cells is presented. The biflagellar green microalga Haematococcus pluvialis was confined with picoNewton trapping forces in the focal volume of a high numerical aperture objective by near infrared multitraps. Trapping radiation of 100 mW power at the sample was provided by a Nd:YLF laser (1047 nm) operating in the cw mode. Simultaneously, cellular autofluorescence was excited with a 633 nm picosecond 80 MHz laser diode. An ultrafast gated intensified slow scan CCD camera system with a tunable gate width (200 ps-1 ms) and tunable time-delay (0-20 ns) between excitation and detection was used as fluorescence detector. We demonstrate fluorescence imaging of high temporal (sub-ns) and high spatial (sub-microm) resolution and fluorescence lifetime determination of intracellular autofluorescence based on chlorophyll excitation. Exposure to the herbicide DCMU resulted in an increase of fluorescence intensity and lifetime by 250% and 150%, respectively.

Chlorophyta↗

PubMed1997

Microtubule-associated protein 2c reorganizes both microtubules and microfilaments into distinct cytological structures in an actin-binding protein-280-deficient melanoma cell line.

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.

Actin Cytoskeleton↗

PubMed1997

Progressive motor neuron impairment in an animal model of familial amyotrophic lateral sclerosis.

Mutations of Cu,Zn superoxide dismutase cause an autosomal dominant form of familial amyotrophic lateral sclerosis. An animal model of the disease has been produced by expressing mutant human SOD1 in transgenic mice (G93A). In order to quantify the dysfunction of the motor unit in transgenic mice, electromyographic recordings were performed during the course of the disease. The first alterations in neuromuscular function appeared between P63 and P90. The deficits became even more striking after P100; compound muscle action potentials in the hindlimb decreased by 80% of initial value. Spontaneous fibrillation potentials were measured in more than 50% of transgenic mice. The number of motor units in the gastrocnemius muscle was progressively reduced over time, down to 18% of the control value at P130. Moreover, distal motor latencies increased after P120. These data suggest that the initial dysfunctions of motor unit are related to a severe motor axonal degeneration, which is followed at later periods by myelin alteration.

Amyotrophic Lateral Sclerosis↗

PubMed1997

I kappaB alpha physically interacts with a cytoskeleton-associated protein through its signal response domain.

The I kappaB alpha protein is a key molecular target involved in the control of NF-kappaB/Rel transcription factors during viral infection or inflammatory reactions. This NF-kappaB-inhibitory factor is regulated by posttranslational phosphorylation and ubiquitination of its amino-terminal signal response domain that targets I kappaB alpha for rapid proteolysis by the 26S proteasome. In an attempt to identify regulators of the I kappaB alpha inhibitory activity, we undertook a yeast two-hybrid genetic screen, using the amino-terminal end of I kappaB alpha as bait, and identified 12 independent interacting clones. Sequence analysis identified some of these cDNA clones as Dlc-1, a sequence encoding a small, 9-kDa human homolog of the outer-arm dynein light-chain protein. In the two-hybrid assay, Dlc-1 also interacted with full-length I kappaB alpha protein but not with N-terminal-deletion-containing versions of I kappaB alpha. I kappaB alpha interacted in vitro with a glutathione S-transferase-Dlc-1 fusion protein, and RelA(p65) did not displace this association, demonstrating that p65 and Dlc-1 contact different protein motifs of I kappaB alpha. Importantly, in HeLa and 293 cells, endogenous and transfected I kappaB alpha coimmunoprecipitated with Myc-tagged or endogenous Dlc-1. Indirect immunofluorescence analyzed by confocal microscopy indicated that Dlc-1 and I kappaB alpha colocalized with both nuclear and cytoplasmic distribution. Furthermore, Dlc-1 and I kappaB alpha were found to associate with the microtubule organizing center, a perinuclear region from which microtubules radiate. Likewise, I kappaB alpha colocalized with alpha-tubulin filaments. Taken together, these results highlight an intriguing interaction between the I kappaB alpha protein and the human homolog of a member of the dynein family of motor proteins and provide a potential link between cytoskeleton dynamics and gene regulation.

Amino Acid Sequence↗