Microtubule depolymerization inhibits transport of cathepsin D from the Golgi apparatus to lysosomes

1990 ◽  
Vol 96 (4) ◽  
pp. 711-720
Author(s):  
J. Scheel ◽  
R. Matteoni ◽  
T. Ludwig ◽  
B. Hoflack ◽  
T.E. Kreis
Keyword(s):  
Golgi Apparatus ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Spatial Organization ◽  
Microtubule Depolymerization ◽  
Microtubule Organizing Center ◽  
Microtubule Network ◽  
Organizing Center ◽  
Efficient Transport ◽  
Golgi Network

Lysosomes as well as a prelysosomal compartment rich in the mannose 6-phosphate receptor are clustered close to the Golgi apparatus in the perinuclear region of the microtubule organizing center in interphase human skin fibroblasts. The spatial organization of these organelles depends on an intact microtubule network. Depolymerization of the microtubules by treatment of cells with nocodazole leads to random scattering of Golgi elements, the prelysosomal compartment, and lysosomes throughout the cytoplasm. To test whether microtubules and the spatial organization of these organelles are important for efficient transport of lysosomal enzymes, the effect of microtubule depolymerization on the maturation of newly synthesized cathepsin D was studied. An up to fivefold inhibition of proteolytic maturation of cathepsin D was observed in drug-treated cells. This effect was due to a decreased rate of transport of cathepsin D from the Golgi apparatus to lysosomes. Depolymerization of microtubules did not inhibit transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi network. Furthermore, synthesis of the phosphomannosyl marker present on cathepsin D was not affected by nocodazole. These results suggest that efficient transport of cathepsin D from the Golgi apparatus to a prelysosomal compartment and lysosomes is facilitated by microtubules and the spatial organization of these organelles.

Effect of shear stress upon localization of the Golgi apparatus and microtubule organizing center in isolated cultured endothelial cells

1993 ◽  
Vol 104 (4) ◽  
pp. 1145-1153 ◽  
Author(s):  
D.E. Coan ◽  
A.R. Wechezak ◽  
R.F. Viggers ◽  
L.R. Sauvage
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Cell Migration ◽  
Golgi Apparatus ◽  
Flow Direction ◽  
Microtubule Organizing Center ◽  
Directed Cell Migration ◽  
Time Period ◽  
Significant Difference ◽  
Organizing Center

Despite substantial evidence to suggest that directed cell migration is dependent upon positioning of the Golgi apparatus (GA) and the microtubule organizing center (MTOC), some controversy exists about whether such a relationship is relevant to endothelial cells under flow. The present study was undertaken to provide an indepth investigation of the relationship between shear stress, GA/MTOC localization, cell migration and nuclear position. Bovine carotid endothelial cells were exposed to 22 or 88 dynes/cm2 for 0.5, 2, 8 or 24 h, and localization of their GA/MTOCs was determined relative to the direction of flow. In no-flow control specimens, (0, 0.5, 2, 8 and 24 h) there was no change in the equally distributed GA/MTOCs. In contrast, during the first 8 h at 88 dynes/cm2 and by 2 h at 22 dynes/cm2 there was a significant increase in the number of cells with GA/MTOCs localized upstream to flow direction. The effect was temporary, however, and by 24 h there was no significant difference between the no-flow, 22 and 88 dynes/cm2 specimens. Analysis of GA/MTOC localization with respect to the direction of cell migration determined that 72.5% of no-flow cells possessed GA/MTOCs localized to the sides of nuclei nearest the direction of migration. In contrast, 64% of the specimens shear stressed over the same time period had GA/MTOCs localized to the sides of nuclei opposite the direction of migration. These results suggest that positioning of the GA/MTOC in endothelial cells is not dependent completely upon the direction of migration.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl- secretion in epithelial cells

1994 ◽  
Vol 266 (1) ◽  
pp. C254-C268 ◽  
Author(s):  
A. P. Morris ◽  
S. A. Cunningham ◽  
A. Tousson ◽  
D. J. Benos ◽  
R. A. Frizzell
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Brefeldin A ◽  
Microtubule Organizing Center ◽  
Cl Secretion ◽  
Organizing Center ◽  
Reversible Time ◽  
Golgi Network ◽  
Ht 29 ◽  
Cl Conductance

The relationship between adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and the cellular location of the cystic fibrosis transmembrane conductance regulator (CFTR) was determined in both polarized (Cl.19A) and unpolarized (parental) HT-29 colonocytes expressing similar levels of CFTR mRNA and protein. CFTR immunolocalized to the apical membrane domain of polarized colonocytes exhibiting cAMP-responsive Cl- secretion. In contrast, CFTR staining was perinuclear in unpolarized colonocytes, which gave little or no cAMP-stimulated Cl- conductance responses. Thus cAMP-stimulated Cl- secretion coincided with an apical localization of CFTR. Brefeldin A (BFA) was used to perturb glycoprotein targeting in these cells. In polarized colonocytes, BFA caused a reversible, time-dependent decrease in the Cl-conductance response to cAMP but not Ca2+. Apical CFTR redistributed into large coalesced intracellular vesicles, located within the same plane as the microtubule organizing center, a marker for the trans-Golgi network (TGN). In preconfluent monolayers or unpolarized HT-29 cells, BFA had no effect on CFTR staining, which remained perinuclear. Mature, Golgi-processed CFTR protein was isolated from both polarized and unpolarized colonocytes. Thus the mechanism for polarization-dependent apical membrane CFTR targeting and the acquisition of cAMP-dependent Cl- secretion lies at or beyond the late Golgi-TGN in epithelial cells.

scholarly journals Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38.

1992 ◽  
Vol 116 (1) ◽  
pp. 85-94 ◽  
Author(s):  
B Reaves ◽  
G Banting
Keyword(s):  
Membrane Protein ◽  
Morphological Changes ◽  
Brefeldin A ◽  
Integral Membrane Protein ◽  
Polyclonal Antiserum ◽  
Atp Depletion ◽  
Microtubule Organizing Center ◽  
Trans Golgi Network ◽  
Organizing Center ◽  
Golgi Network

Brefeldin A (BFA) has a dramatic effect on the morphology of the Golgi apparatus and induces a rapid redistribution of Golgi proteins into the ER (Lippincott-Schwartz, J., L. C. Yuan, J. S. Bonifacino, and R. D. Klausner. 1989. Cell. 56:801-813). To date, no evidence that BFA affects the morphology of the trans-Golgi network (TGN) has been presented. We describe the results of experiments, using a polyclonal antiserum to a TGN specific integral membrane protein (TGN38) (Luzio, J.P., B. Brake, G. Banting, K. E. Howell, P. Braghetta, and K. K. Stanley. 1990. Biochem. J. 270:97-102), which demonstrate that incubation of cells with BFA does induce morphological changes to the TGN. However, rather than redistributing to the ER, the majority of the TGN collapses around the microtubule organizing center (MTOC). The effect of BFA upon the TGN is (a) independent of protein synthesis, (b) fully reversible (c) microtubule dependent (as shown in nocodazole-treated cells), and (d) relies upon the hydrolysis of GTP (as shown by performing experiments in the presence of GTP gamma S). ATP depletion reduces the ability of BFA to induce a redistribution of Golgi proteins into the ER; however, it has no effect upon the BFA-induced relocalizations of the TGN. These data confirm that the TGN is an organelle which is independent of the Golgi, and suggest a dynamic interaction between the TGN and microtubules which is centered around the MTOC.

scholarly journals Inhibition by cyanate of the processing of lysosomal enzymes

1983 ◽  
Vol 210 (3) ◽  
pp. 795-802 ◽  
Author(s):  
A Hasilik ◽  
R Pohlmann ◽  
K von Figura
Keyword(s):  
Golgi Apparatus ◽  
Lysosomal Enzyme ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Human Fibroblasts ◽  
Cultured Human Fibroblasts ◽  
High Mannose ◽  
In Cells

In cultured human fibroblasts, maturation of the lysosomal enzymes beta-hexosaminidase and cathepsin D is inhibited by 10 mM-potassium cyanate. In cells treated with cyanate the two enzymes accumulate in precursor forms. The location of the accumulated precursor is probably non-lysosomal; in fractionation experiments the precursors separate from the bulk of the beta-hexosaminidase activity. The secretion of the precursor of cathepsin D, but not that of beta-hexosaminidase precursor, is enhanced in the presence of cyanate. The secreted cathepsin D, as well as that remaining within the cells, contains mostly high-mannose oligosaccharides cleavable with endo-beta-N-acetylglucosaminidase H. After removal of cyanate, the accumulated precursor forms of the lysosomal enzymes are largely released from the pretreated cells. It is concluded that cyanate interferes with the maturation of lysosomal-enzyme precursors by perturbing their intracellular transport. Most probably cyanate affects certain functions of the Golgi apparatus.

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 Mutations in the Amino Terminus of Foamy Virus Gag Disrupt Morphology and Infectivity but Do Not Target Assembly

2008 ◽  
Vol 82 (13) ◽  
pp. 6109-6119 ◽  
Author(s):  
Rachel B. Life ◽  
Eun-Gyung Lee ◽  
Scott W. Eastman ◽  
Maxine L. Linial
Keyword(s):  
Plasma Membrane ◽  
Foamy Virus ◽  
Amino Terminus ◽  
Microtubule Organizing Center ◽  
Interaction Domain ◽  
Target Assembly ◽  
Organizing Center ◽  
Morphological Defects ◽  
Bona Fide ◽  
Golgi Network

ABSTRACT Foamy viruses (FVs) assemble using pathways distinct from those of orthoretroviruses. FV capsid assembly takes place near the host microtubule-organizing center (MTOC). Assembled capsids then migrate by an unknown mechanism to the trans-Golgi network to colocalize with the FV glycoprotein, Env. Interaction with Env is required for FV capsid egress from cells; the amino terminus of FV Gag contains a cytoplasmic targeting/retention signal that is responsible for targeting assembly to the MTOC. A mutant Gag was constructed by addition of a myristylation (M) signal in an attempt to target assembly to the plasma membrane and potentially overcome the dependence upon Env for budding (S. W. Eastman and M. L. Linial, J. Virol. 75:6857-6864, 2001). Using this and additional mutants, we now show that assembly is not redirected to the plasma membrane. Addition of an M signal leads to gross morphological defects. The aberrant particles still assemble near the MTOC but do not produce infectious virus. Although extracellular Gag can be detected in a pelletable form in the absence of Env, the mutant particles contain very little genomic RNA and are less dense. Our analyses indicate that the amino terminus of Gag contains an Env interaction domain that is critical for bona fide egress of assembled capsids.

scholarly journals Microtubules control cellular shape and coherence in amoeboid migrating cells

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Aglaja Kopf ◽  
Jörg Renkawitz ◽  
Robert Hauschild ◽  
Irute Girkontaite ◽  
Kerry Tedford ◽  
...  
Keyword(s):  
Cell Shape ◽  
Shape Control ◽  
Microtubule Depolymerization ◽  
Microtubule Organizing Center ◽  
Amoeboid Cell ◽  
Actomyosin Contractility ◽  
Organizing Center ◽  
A Cell ◽  
And Control ◽  
Rate Limit

Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence.

The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A

1993 ◽  
Vol 106 (3) ◽  
pp. 789-802 ◽  
Author(s):  
M. Roa ◽  
V. Cornet ◽  
C.Z. Yang ◽  
B. Goud
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Brefeldin A ◽  
Atp Depletion ◽  
Cell Fractionation ◽  
Microtubule Organizing Center ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Organizing Center ◽  
Golgi Network

Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10–15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center. The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence. Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.

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