scholarly journals Developmental-Dependent Action of Microtubule Depolymerization on the Function and Structure of Synaptic Glycine Receptor Clusters in Spinal Neurons

2004 ◽  
Vol 91 (2) ◽  
pp. 1036-1049 ◽  
Author(s):  
Brigitte van Zundert ◽  
Francisco J. Alvarez ◽  
Juan Carlos Tapia ◽  
Hermes H. Yeh ◽  
Emilio Diaz ◽  
...  
Keyword(s):  
Average Length ◽  
Glycine Receptor ◽  
Colchicine Treatment ◽  
Morphological Properties ◽  
Microtubule Depolymerization ◽  
Spinal Neurons ◽  
Functional Changes ◽  
Microtubule Disruption ◽  
Immature Neurons ◽  
Mature Neurons

Microtubules have been proposed to interact with gephyrin/glycine receptors (GlyRs) in synaptic aggregates. However, the consequence of microtubule disruption on the structure of postsynaptic GlyR/gephyrin clusters is controversial and possible alterations in function are largely unknown. In this study, we have examined the physiological and morphological properties of GlyR/gephyrin clusters after colchicine treatment in cultured spinal neurons during development. In immature neurons (5-7 DIV), disruption of microtubules resulted in a 33 ± 4% decrease in the peak amplitude and a 72 ± 15% reduction in the frequency of spontaneous glycinergic miniature postsynaptic currents (mIPSCs) recorded in whole cell mode. However, similar colchicine treatments resulted in smaller effects on 10-12 DIV neurons and no effect on mature neurons (15-17 DIV). The decrease in glycinergic mIPSC amplitude and frequency reflects postsynaptic actions of colchicine, since postsynaptic stabilization of microtubules with GTP prevented both actions and similar reductions in mIPSC frequency were obtained by modifying the Cl- driving force to obtain parallel reductions in mIPSC amplitude. Confocal microscopy revealed that colchicine reduced the average length and immunofluorescence intensity of synaptic gephyrin/GlyR clusters in immature (approximately 30%) and intermediate (approximately 15%) neurons, but not in mature clusters. Thus the structural and functional changes of postsynaptic gephyrin/GlyR clusters after colchicine treatment were tightly correlated. Finally, RT-PCR, kinetic analysis and picrotoxin blockade of glycinergic mIPSCs indicated a reorganization of the postsynaptic region from containing both α2β and α1β GlyRs in immature neurons to only α1β GlyRs in mature neurons. Microtubule disruption preferentially affected postsynaptic sites containing α2β-containing synaptic receptors.

Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells

1991 ◽  
Vol 261 (1) ◽  
pp. C691-C700 ◽  
Author(s):  
J. B. De Almeida ◽  
J. L. Stow
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Membrane Protein ◽  
Basolateral Membrane ◽  
Colchicine Treatment ◽  
Immunofluorescent Staining ◽  
Secretory Proteins ◽  
Microtubule Depolymerization ◽  
Microtubule Network ◽  
Microtubule Disruption

Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na(+)-K(+)-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface.

scholarly journals THE EFFECT OF COLCHICINE ON MYOGENESIS IN VIVO IN RANA PIPIENS AND RHODNIUS PROLIXUS (HEMIPTERA)

1968 ◽  
Vol 39 (3) ◽  
pp. 544-555 ◽  
Author(s):  
Robert H. Warren
Keyword(s):  
Cell Shape ◽  
Rana Pipiens ◽  
Colchicine Treatment ◽  
Rhodnius Prolixus ◽  
Microtubule Disruption ◽  
Blood Sucking ◽  
Tadpole Tail ◽  
Differentiation And Dedifferentiation ◽  
Regeneration Blastema

The effect of colchicine on myogenesis in vivo has been studied in the regenerating tadpole tail of the frog, Rana pipiens, and in the abdominal molting muscles of a blood-sucking bug, Rhodnius prolixus Stål. Colchicine is shown to disrupt microtubules in the differentiating muscle cells of both these organisms. The disruption of microtubules is correlated with a loss of longitudinal anisometry in the myoblasts and myotubes of the regeneration blastema in the tadpole tail. Before colchicine treatment, the myotubes contain longitudinally oriented myofibrils. After colchicine treatment, rounded, multinucleate myosacs containing randomly oriented myofibrils are present. It is suggested that the primary function of microtubules in myogenesis in the Rana pipiens tadpole is the maintenance of cell shape. The abdominal molting muscles of Rhodnius undergo repeated phases of differentiation and dedifferentiation of the sarcoplasm. However, the longitudinal anisometry of the muscle fibers is maintained in all phases by the attachments of the ends of the fibers to the exoskeleton, and microtubule disruption does not alter cell shape. The orientation of the developing myofibrils is also unaltered, indicating that the microtubules do not directly align or support the myofibrils in this system.

Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells

1991 ◽  
Vol 260 (4) ◽  
pp. C691-C700 ◽  
Author(s):  
J. Bruno de Almeida ◽  
Jennifer L. Stow
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Membrane Protein ◽  
Heparan Sulfate ◽  
Basolateral Membrane ◽  
Immunofluorescent Staining ◽  
Secretory Proteins ◽  
Microtubule Depolymerization ◽  
Microtubule Network ◽  
Microtubule Disruption

Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na+-K+-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface. polarized secretion; heparan sulfate proteoglycans; sorting; Golgi processing Submitted on July 10, 1990 Accepted on November 12, 1990

scholarly journals NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

2019 ◽  
Vol 93 (24) ◽  
Author(s):  
Jane X. Yeh ◽  
Eunhye Park ◽  
Kimberly L. W. Schultz ◽  
Diane E. Griffin
Keyword(s):  
Protein Synthesis ◽  
Neuronal Cells ◽  
Structural Proteins ◽  
Neuronal Maturation ◽  
Viral Encephalomyelitis ◽  
Pathway Activation ◽  
Age Dependent ◽  
Immature Neurons ◽  
Mature Neurons

ABSTRACT Alphaviruses are enveloped, positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) infects the neurons of rodents and is a model for studying factors that regulate infection of neuronal cells. The outcome of alphavirus infection of the central nervous system is dependent on neuronal maturation status. Differentiated mature neurons survive and control viral replication better than undifferentiated immature neurons. The cellular factors involved in age-dependent susceptibility include higher levels of antiapoptotic and innate immune factors in mature neurons. Because NF-κB pathway activation is required for the initiation of both apoptosis and the host antiviral response, we analyzed the role of NF-κB during SINV infection of differentiated and undifferentiated rat neuronal cells. SINV infection induced canonical NF-κB activation, as evidenced by the degradation of IκBα and the phosphorylation and nuclear translocation of p65. Inhibition or deletion of the upstream IκB kinase substantially reduced SINV replication in differentiated but not in undifferentiated neuronal cells or mouse embryo fibroblasts. NF-κB inhibition did not affect the establishment of infection, replication complex formation, the synthesis of nonstructural proteins, or viral RNA synthesis in differentiated neurons. However, the translation of structural proteins was impaired, phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) was decreased, and host protein synthesis was maintained, suggesting that NF-κB activation was involved in the regulation of translation during infection of mature neurons. Inhibition or deletion of double-stranded RNA-activated protein kinase (PKR) also decreased eIF2α phosphorylation, the translation of viral structural proteins, and virus production. Therefore, canonical NF-κB activation synergizes with PKR to promote SINV replication in differentiated neurons by facilitating viral structural protein translation. IMPORTANCE Mosquito-borne alphaviruses are a significant and growing cause of viral encephalomyelitis worldwide. The outcome of alphaviral neuronal infections is host age dependent and greatly affected by neuronal maturation status, with differentiated, mature neurons being more resistant to infection than undifferentiated, immature neurons. The biological factors that change during neuronal maturation and that influence the outcome of viral infection are currently only partially defined. These studies investigated the role of NF-κB in determining the outcome of alphaviral infection in mature and immature neurons. Inhibition of canonical NF-κB activation decreased alphavirus replication in mature neurons by regulating protein synthesis and limiting the production of the viral structural proteins but had little effect on viral replication in immature neurons or fibroblasts. Therefore, NF-κB is a signaling pathway that influences the maturation-dependent outcome of alphaviral infection in neurons and that highlights the importance of cellular context in determining the effects of signal pathway activation.

Microtubules, colchicine, and lymphocyte blastogenesis

1979 ◽  
Vol 57 (6) ◽  
pp. 673-683 ◽  
Author(s):  
Christopher E. Rudd ◽  
Kem A. Rogers ◽  
David L. Brown ◽  
J. Gordin Kaplan
Keyword(s):  
Dna Synthesis ◽  
Time Course ◽  
Human Lymphocytes ◽  
Colchicine Treatment ◽  
Con A ◽  
Lymphocyte Blastogenesis ◽  
Dna And Rna ◽  
Microtubule Disruption ◽  
Inhibition Of Dna Synthesis ◽  
Mouse Lymphocytes

We have studied the time course of disassembly of microtubules of resting and stimulated mouse lymphocytes caused by the drug colchicine, as well as the effect of this compound on DNA and RNA synthesis of human and mouse lymphocytes. Fine-structure studies with the electron microscope showed a great increase in number of microtubules resulting from stimulation of mouse lymphocytes by the mitogenic lectin Con A. The presence of a network of microtubules was demonstrated in resting lymphocytes by use of the technique of immunofluorescence; this technique was not effective for the study of the microtubules of stimulated lymphocytes in the blast stage. The disappearance of microtubular networks in some cells (approximately 25%) was caused by the protocol of colchicine treatment used in many laboratories (30 min at 10−6 M); a 6- to 8-h treatment was required to cause all cells to lose their microtubules. It is indicated in these findings that there is need for extreme caution in implicating microtubule disruption as the cause of certain colchicine effects, such as that on the Con A-induced inhibition of receptor–ligand migration. The addition of colchicine to stimulated cells at varying times of culture caused marked inhibition of DNA synthesis provided that sufficient time (approximately 20 h for maximum inhibition) elapsed between addition of the drug to the stimulated culture and assay of DNA synthesis. Our data on the time course of inhibition of DNA synthesis by α-methyl mannoside (αMM) and by colchicine do not exclude the possibility that the latter compound may act partly by affecting the commitment of stimulated lymphocytes to DNA synthesis but they show that it can inhibit well after commitment is complete. The later the time of assay of thymidine incorporation, the more disparate were the curves relating the effects of αMM and colchicine to DNA synthesis of human cells. In the case of mouse splenic lymphocytes, there was no resemblance between the time course of the αMM and of the colchicine effects. Synthesis of RNA after 12 h of culture of stimulated human lymphocytes was also sensitive to colchicine.

scholarly journals The Golgi apparatus remains associated with microtubule organizing centers during myogenesis.

1985 ◽  
Vol 101 (2) ◽  
pp. 630-638 ◽  
Author(s):  
A M Tassin ◽  
M Paintrand ◽  
E G Berger ◽  
M Bornens
Keyword(s):  
Golgi Apparatus ◽  
Nuclear Periphery ◽  
Spatial Relationship ◽  
Cell Types ◽  
Microtubule Depolymerization ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Microtubule Disruption ◽  
Tight Association

In vitro myogenesis involves a dramatic reorganization of the microtubular network, characterized principally by the relocalization of microtubule nucleating sites at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization observed in myoblasts (Tassin, A. M., B. Maro, and M. Bornens, 1985, J. Cell Biol., 100:35-46). Since a spatial relationship between the Golgi apparatus and the centrosome is observed in most animal cells, we have decided to follow the fate of the Golgi apparatus during myogenesis by an immunocytochemical approach, using wheat germ agglutinin and an affinity-purified anti-galactosyltransferase. We show that Golgi apparatus in myotubes displays a perinuclear distribution which is strikingly different from the polarized juxtanuclear organization observed in myoblasts. As a result, the Golgi apparatus in myotubes is situated close to the microtubule organizing center (MTOC), the cis-side being situated at a fixed distance from the nuclear envelope, a situation which suggests the existence of a structural association between the Golgi apparatus and the nuclear periphery. This is supported by experiments of microtubule depolymerization by nocodazole, in which a minimal effect was observed on Golgi apparatus localization in myotubes in contrast with the dramatic scattering observed in myoblasts. In both cell types, electron microscopy reveals that microtubule disruption generates individual dictyosomes; this suggests that the connecting structures between dictyosomes are principally affected. This structural dependency of the Golgi apparatus upon microtubules is not apparently accompanied by a reverse dependency of MTOC structure or function upon Golgi apparatus activity. Golgi apparatus modification by monensin, as effective in myotubes as in myoblasts, is without apparent effect on MTOC localization or activity and on microtubule stability. The main result of our study is to show that in a cell type where the MTOC is dissociated from centrioles and where antero-posterior polarity has disappeared, the association between the Golgi apparatus and the MTOC is maintained. The significance of such a tight association is discussed.

scholarly journals Phylogenetic variation in cortical layer II immature neuron reservoir of mammals

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Chiara La Rosa ◽  
Francesca Cavallo ◽  
Alessandra Pecora ◽  
Matteo Chincarini ◽  
Ugo Ala ◽  
...  
Keyword(s):  
Brain Size ◽  
Mammalian Species ◽  
Cortical Layer ◽  
Mammalian Brain ◽  
Postnatal Life ◽  
Molecular Features ◽  
Immature Neuron ◽  
Immature Neurons ◽  
Mature Neurons ◽  
Adult Mammalian Brain

The adult mammalian brain is mainly composed of mature neurons. A limited amount of stem cell-driven neurogenesis persists in postnatal life and is reduced in large-brained species. Another source of immature neurons in adult brains is cortical layer II. These cortical immature neurons (cINs) retain developmentally undifferentiated states in adulthood, though they are generated before birth. Here, the occurrence, distribution and cellular features of cINs were systematically studied in 12 diverse mammalian species spanning from small-lissencephalic to large-gyrencephalic brains. In spite of well-preserved morphological and molecular features, the distribution of cINs was highly heterogeneous, particularly in neocortex. While virtually absent in rodents, they are present in the entire neocortex of many other species and their linear density in cortical layer II generally increased with brain size. These findings suggest an evolutionary developmental mechanism for plasticity that varies among mammalian species, granting a reservoir of young cells for the cerebral cortex.

scholarly journals Optogenetic silencing of immature and mature neurons in dentate gyrus to assess their roles in memory discriminations

2018 ◽  
Author(s):  
Milenna T. van Dijk ◽  
Zejia Angel Yu ◽  
Younghun Lim ◽  
René Hen ◽  
André A. Fenton
Keyword(s):  
Dentate Gyrus ◽  
Granule Cells ◽  
Prior Experience ◽  
Immature Neurons ◽  
Mature Neurons ◽  
Place Avoidance ◽  
Dentate Gyrus Region

ABSTRACTDiscriminating similar memories and events depends on the dentate gyrus region of the hippocampus. This region is also distinctive because neurogenesis continues in adulthood. Whether both mature and immature granule cells play a role in memory discrimination, and whether the roles are distinct is actively investigated. Here we demonstrate that manipulating either mature or immature granule cells can impair discrimination of similar active place avoidance memories, but the manipulations have different effects. We also observe that prior experience modulates which memories are compromised by inactivation of immature neurons. These data demonstrate the importance of the dentate gyrus network of cells for memory discrimination.

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