scholarly journals Mitotic disassembly of the Golgi apparatus in vivo

1995 ◽  
Vol 108 (7) ◽  
pp. 2715-2727 ◽  
Author(s):  
T. Misteli ◽  
G. Warren
Keyword(s):  
Golgi Apparatus ◽  
Morphological Changes ◽  
Temperature Sensitive ◽  
Cross Sectional ◽  
Mitotic Kinase ◽  
Transport Vesicles ◽  
A Cell ◽  
Cell Cycle Block ◽  
Conventional Electron Microscopy

Populations enriched in prophase cells were obtained either by using a cell line with a temperature-sensitive mutation in the mitotic kinase, p34cdc2, or by treating cells with olomoucine, an inhibitor of this kinase. Both methods resulted in efficient and reversible block of the cells at the G2/M boundary. After cells were released from the cell cycle block, the morphological changes to the Golgi apparatus were characterised using both quantitative conventional electron microscopy and immuno-gold microscopy. The early mitotic phases were divided into six stages (G2 to pro-metaphase) based on the morphology of the nucleus. During prophase the cross-sectional length of Golgi stacks decreased prior to unstacking. At the same time, small vesicular profiles, typically 50–70 nm in diameter, accumulated in the vicinity of the stacks. The disappearance of Golgi stacks was accompanied by the transient appearance of tubular networks. By the time cells entered prometaphase, the stacks had completely disassembled and only clusters consisting of Golgi vesicles and short tubular elements were left. When cells were released from the G2/M boundary and pulsed briefly with [AlF4]- to prevent uncoating of transport vesicles, vesicular profiles with a morphology reminiscent of COP-coated vesicles appeared. These vesicular profiles were either associated with Golgi stacks or, at later stages, with clusters, but were formed at all stages of disassembly. Together these results provide further support for our model that continued budding of vesicles from the rims of Golgi cisternae is at least partly responsible for the disassembly of the Golgi apparatus.

Dynamics of the Tracheal Airway and Its Influences on Respiratory Airflows: An Exemplar Study

2019 ◽  
Vol 141 (11) ◽  
Author(s):  
Bora Sul ◽  
Talissa Altes ◽  
Kai Ruppert ◽  
Kun Qing ◽  
Daniel S. Hariprasad ◽  
...  
Keyword(s):  
Morphological Changes ◽  
Minute Ventilation ◽  
Correlation Coefficients ◽  
Dynamic Imaging ◽  
Imaging Data ◽  
Cross Sectional ◽  
Mild Conditions ◽  
Large Airways ◽  
Magnetic Resonance Imaging Mri

Respiration is a dynamic process accompanied by morphological changes in the airways. Although deformation of large airways is expected to exacerbate pulmonary disease symptoms by obstructing airflow during increased minute ventilation, its quantitative effects on airflow characteristics remain unclear. Here, we used in vivo dynamic imaging and examined the effects of tracheal deformation on airflow characteristics under different conditions based on imaging data from a single healthy volunteer. First, we measured tracheal deformation profiles of a healthy lung using magnetic resonance imaging (MRI) during forced exhalation, which we simulated to characterize the subject-specific airflow patterns. Subsequently, for both inhalation and exhalation, we compared the airflows when the modeled deformation in tracheal cross-sectional area was 0% (rigid), 33% (mild), 50% (moderate), or 75% (severe). We quantified differences in airflow patterns between deformable and rigid airways by computing the correlation coefficients (R) and the root-mean-square of differences (Drms) between their velocity contours. For both inhalation and exhalation, airflow patterns were similar in all branches between the rigid and mild conditions (R > 0.9; Drms < 32%). However, airflow characteristics in the moderate and severe conditions differed markedly from those in the rigid and mild conditions in all lung branches, particularly for inhalation (moderate: R > 0.1, Drms < 76%; severe: R > 0.2, Drms < 96%). Our exemplar study supports the use of a rigid airway assumption to compute flows for mild deformation. For moderate or severe deformation, however, dynamic contraction should be considered, especially during inhalation, to accurately predict airflow and elucidate the underlying pulmonary pathology.

scholarly journals Leukemic cell differentiation in vivo and in vitro: arrest of proliferation parallels the differentiation induced by the antileukemic drug Harringtonine

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 384-392 ◽  
Author(s):  
AW Boyd ◽  
JR Sullivan
Keyword(s):  
Morphological Changes ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Neoplastic Cell ◽  
Morphological Criteria ◽  
Antileukemic Effect ◽  
Blast Cells ◽  
A Cell

Abstract A variety of chemical agents have been shown to induce differentiation in in vitro cultured neoplastic cell lines. We noted that blast cells in the peripheral blood of acute nonlymphoid leukemia patients treated with the drug Harringtonine appeared to undergo morphological changes that suggested differentiation. In view of the relatively minimal myelotoxicity of Harringtonine, we hypothesized that harringtonine was acting by differentiation-induction, which concomitantly arrested cell division. We tested this hypothesis using two different experimental approaches. First, the promyelocytic leukemic cell line, HL-60, was cultured with Harringtonine and shown to differentiate into a cell, which, by functional cell surface marker and morphological criteria, closely resembled normal monocytes. Furthermore, these changes were accompanied, and indeed slightly preceded by, loss of proliferative capacity. Second, to prove that the leukemic blasts were the cells undergoing the changes observed in vivo, freshly isolated leukemia cell populations were cultured with Harringtonine, and morphological changes paralleling those seen in the patients were observed. Thus, the antileukemic effect of Harringtonine appeared to be due to diversion of the proliferating blast cells into a differentiation pathway, which, as in normal myeloid cells, resulted in the arrest of proliferation.

scholarly journals EFFECTS OF PHOSPHOTUNGSTATE NEGATIVE STAINING ON THE MORPHOLOGY OF THE ISOLATED GOLGI APPARATUS

1974 ◽  
Vol 62 (2) ◽  
pp. 491-504 ◽  
Author(s):  
William P. Cunningham ◽  
L. Andrew Staehelin ◽  
Robert W. Rubin ◽  
Ross Wilkins ◽  
Mary Bonneville
Keyword(s):  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Protein Extraction ◽  
Membrane Lipids ◽  
Morphological Changes ◽  
Negative Staining ◽  
Thin Sections ◽  
Whole Cells ◽  
Negative Stain

Isolated Golgi complexes can be recognized in phosphotungstate (PTA) negative stain as stacks of membranous plates surrounded by a complex anastomosing network of tubules and vesicles. The extent of this tubular network is, however, much greater than can be observed in thin sections of whole cells. To determine which of the steps leading to the final negatively stained image may produce the observed changes, we have monitored each of the steps by other electron microscope and biochemical methods. The first damage to the membranes seems to occur during the initial isolation procedure as judged by the appearance of smooth patches on the freeze-fractured membrane faces that are normally covered with particles. Subsequent suspension of the Golgi fraction in water, to dilute the sucrose for negative staining, leads to the disappearnce of the stacking, to some tubulation and some vesiculation of the membranes as judged by thin section and freeze-cleave microscopy. The latter technique also reveals an increase in smooth-cleaving membrane faces. Application of the negative stain to the water-washed Golgi fraction, finally, produces extensive tubular arrays and a simultaneous decrease in the remaining large membranous vesicles. The freeze-cleaved tubular membranes appear essentially smooth except for small patches of aggregated particles. Parallel gel electrophoresis studies of the membranes and of the water and negative stain wash extracts indicate that protein extraction is involved in these morphological changes. PTA seems to be a particularly effective solvent for certain membrane proteins that are not removed by the water wash. These observations suggest that removal of membrane proteins alters structural restraints on the membrane lipids so that they behave semiautonomously like myelinics and form new artificial structures. This does not eliminate the possibility, however, that some tubules also exist in the Golgi apparatus in vivo.

scholarly journals COP-coated vesicles are involved in the mitotic fragmentation of Golgi stacks in a cell-free system.

1994 ◽  
Vol 125 (2) ◽  
pp. 269-282 ◽  
Author(s):  
T Misteli ◽  
G Warren
Keyword(s):  
Strong Support ◽  
Serial Sectioning ◽  
Animal Cells ◽  
Cdc2 Kinase ◽  
Cross Sectional ◽  
Free System ◽  
Transport Vesicles ◽  
A Cell ◽  
Gamma S ◽  
Total Membrane

Rat liver Golgi stacks fragmented when incubated with mitotic but not interphase cytosol in a process dependent on time, temperature, energy (added in the form of ATP) and cdc2 kinase. The cross-sectional length of Golgi stacks fell in the presence of mitotic cytosol by approximately 50% over 30 min without a corresponding decrease in the number of cisternae in the stack. The loss of membrane from stacked and single cisternae occurred with a half-time of approximately 20 min, and was matched by the appearance of both small (50-100 nm in diameter) and large (100-200 nm in diameter) vesicular profiles. Small vesicular profiles constituted more than 50% of the total membrane after 60 min of incubation and they were shown to be vesicles or very short tubules by serial sectioning. In the presence of GTP gamma S all of the small vesicles were COP-coated and both the extent and the rate at which they formed were sufficient to account for the production of small vesicles during mitotic incubation. The involvement of the COP-mediated budding mechanism was confirmed by immunodepletion of one of the subunits of COP coats (the coatomer) from mitotic cytosol. Vesicles were no longer formed but highly fenestrated networks appeared, an effect reversed by the readdition of purified coatomer. Together these experiments provide strong support for our hypothesis that the observed vesiculation of the Golgi apparatus during mitosis in animal cells is caused by continued budding of COP-coated transport vesicles but an inhibition of their fusion with their target membranes.

scholarly journals Dynamin at the Neck of Caveolae Mediates Their Budding to Form Transport Vesicles by GTP-driven Fission from the Plasma Membrane of Endothelium

1998 ◽  
Vol 141 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Phil Oh ◽  
Deirdre P. McIntosh ◽  
Jan E. Schnitzer
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Intact Cell ◽  
Subcellular Fractionation ◽  
Transport Vesicles ◽  
Cell Plasma ◽  
A Cell ◽  
Free Transport

The molecular mechanisms mediating cell surface trafficking of caveolae are unknown. Caveolae bud from plasma membranes to form free carrier vesicles through a “pinching off” or fission process requiring cytosol and driven by GTP hydrolysis (Schnitzer, J.E., P. Oh, and D.P. McIntosh. 1996. Science. 274:239–242). Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems to establish a function for dynamin in the formation of transport vesicles from the endothelial cell plasma membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic dynamin as well as its hydrolysis of GTP. Expression of dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in a cell-free assay whereas its removal from cytosol or the addition to the cytosol of specific antibodies for dynamin inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin B chain in intact and permeabilized endothelial cells. Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is concentrated on caveolae, primarily at the expected site of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar around the neck of caveolae that hydrolyzes GTP to mediate internalization via the fission of caveolae from the plasma membrane to form free transport vesicles.

scholarly journals A Role for Tlg1p in the Transport of Proteins within the Golgi Apparatus ofSaccharomyces cerevisiae

1999 ◽  
Vol 10 (7) ◽  
pp. 2407-2423 ◽  
Author(s):  
John G. S. Coe ◽  
Anthony C. B. Lim ◽  
Jing Xu ◽  
Wanjin Hong
Keyword(s):  
Golgi Apparatus ◽  
Snare Complex ◽  
Growth Defect ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dependent Manner ◽  
Nonpermissive Temperature ◽  
Vacuolar Protein

Members of the syntaxin protein family participate in the docking–fusion step of several intracellular vesicular transport events. Tlg1p has been identified as a nonessential protein required for efficient endocytosis as well as the maintenance of normal levels of trans-Golgi network proteins. In this study we independently describe Tlg1p as an essential protein required for cell viability. Depletion of Tlg1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the early Golgi. Temperature-sensitive (ts) mutants of Tlg1p also accumulate the endoplasmic reticulum/cis-Golgi form of carboxypeptidase Y at the nonpermissive temperature (38°C) and exhibit underglycosylation of secreted invertase. Overexpression of Tlg1p complements the growth defect of vti1-11 at the nonpermissive temperature, whereas incomplete complementation was observed with vti1-1, further suggesting a role for Tlg1p in the Golgi apparatus. Overexpression of Sed5p decreases the viability of tlg1 ts mutants compared with wild-type cells, suggesting that tlg1 ts mutants are more susceptible to elevated levels of Sed5p. Tlg1p is able to bind His6-tagged Sec17p (yeast α-SNAP) in a dose-dependent manner and enters into a SNARE complex with Vti1p, Tlg2p, and Vps45p. Morphological analyses by electron microscopy reveal that cells depleted of Tlg1p or tlg1 ts mutants incubated at the restrictive temperature accumulate 40- to 50-nm vesicles and experience fragmentation of the vacuole.

scholarly journals Leukemic cell differentiation in vivo and in vitro: arrest of proliferation parallels the differentiation induced by the antileukemic drug Harringtonine

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 384-392 ◽  
Author(s):  
AW Boyd ◽  
JR Sullivan
Keyword(s):  
Morphological Changes ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Neoplastic Cell ◽  
Morphological Criteria ◽  
Antileukemic Effect ◽  
Blast Cells ◽  
A Cell

A variety of chemical agents have been shown to induce differentiation in in vitro cultured neoplastic cell lines. We noted that blast cells in the peripheral blood of acute nonlymphoid leukemia patients treated with the drug Harringtonine appeared to undergo morphological changes that suggested differentiation. In view of the relatively minimal myelotoxicity of Harringtonine, we hypothesized that harringtonine was acting by differentiation-induction, which concomitantly arrested cell division. We tested this hypothesis using two different experimental approaches. First, the promyelocytic leukemic cell line, HL-60, was cultured with Harringtonine and shown to differentiate into a cell, which, by functional cell surface marker and morphological criteria, closely resembled normal monocytes. Furthermore, these changes were accompanied, and indeed slightly preceded by, loss of proliferative capacity. Second, to prove that the leukemic blasts were the cells undergoing the changes observed in vivo, freshly isolated leukemia cell populations were cultured with Harringtonine, and morphological changes paralleling those seen in the patients were observed. Thus, the antileukemic effect of Harringtonine appeared to be due to diversion of the proliferating blast cells into a differentiation pathway, which, as in normal myeloid cells, resulted in the arrest of proliferation.

scholarly journals Essential functions of Sds22p in chromosome stability and nuclear localization of PP1

2002 ◽  
Vol 115 (1) ◽  
pp. 195-206 ◽  
Author(s):  
Mark W. Peggie ◽  
Sarah H. MacKelvie ◽  
Andrew Bloecher ◽  
Elena V. Knatko ◽  
Kelly Tatchell ◽  
...  
Keyword(s):  
Chromosome Instability ◽  
Protein Phosphatase 1 ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
A Cell ◽  
Ts Mutant ◽  
Leucine Rich Repeat Protein

Sds22p is a conserved, leucine-rich repeat protein that interacts with the catalytic subunit of protein phosphatase 1 (PP1C) and which has been proposed to regulate one or more functions of PP1C during mitosis. Here we show that Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is present as a sTable 1:1 complex with yeast PP1C (Glc7p). Temperature-sensitive (Ts–) S. cerevisiae sds22 mutants show profound chromosome instability at elevated growth temperatures but do not confer a cell cycle stage-specific arrest. In the sds22-6 Ts– mutant, nuclear Glc7p is both reduced in level and aberrantly localized at 37°C and the interaction between Glc7p and Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo Ts– growth defect. Like some glc7 mutations, sds22-6 can suppress the Ts– growth defect associated with ipl1-2, a loss of function mutation in a protein kinase that is known to work in opposition to PP1 on at least two nuclear substrates. This, together with reciprocal genetic interactions between GLC7 and SDS22, suggests that Sds22p functions positively with Glc7p to promote dephosphorylation of nuclear substrates required for faithful transmission of chromosomes during mitosis, and this role is at least partly mediated by effects of Sds22p on the nuclear distribution of Glc7p

scholarly journals The function of GORASPs in Golgi apparatus organization in vivo

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Rianne Grond ◽  
Tineke Veenendaal ◽  
Juan M. Duran ◽  
Ishier Raote ◽  
Johan H. van Es ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Dynamic Control ◽  
Vesicle Fusion ◽  
In Vitro Experiments ◽  
Cross Sectional ◽  
Data Support

In vitro experiments have shown that GRASP65 (GORASP1) and GRASP55 (GORASP2) proteins function in stacking Golgi cisternae. However, in vivo depletion of GORASPs in metazoans has given equivocal results. We have generated a mouse lacking both GORASPs and find that Golgi cisternae remained stacked. However, the stacks are disconnected laterally from each other, and the cisternal cross-sectional diameters are significantly reduced compared with their normal counterparts. These data support earlier findings on the role of GORASPs in linking stacks, and we suggest that unlinking of stacks likely affects dynamic control of COPI budding and vesicle fusion at the rims. The net result is that cisternal cores remain stacked, but cisternal diameter is reduced by rim consumption.

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