Cytoplasmic assembly of microtubules in cultured cells

1997 ◽  
Vol 110 (21) ◽  
pp. 2635-2645 ◽  
Author(s):  
I.A. Vorobjev ◽  
T.M. Svitkina ◽  
G.G. Borisy
Keyword(s):  
Cultured Cells ◽  
Dynamic Instability ◽  
Time Lapse ◽  
Necessary Condition ◽  
Animal Cells ◽  
Apparent Diffusion Coefficients ◽  
Apparent Diffusion ◽  
Free Microtubules ◽  
Microtubule Formation

The origin of non-centrosomal microtubules was investigated in a variety of animal cells in culture by means of time-lapse digital fluorescence microscopy. A previous study (Keating et al. (1997) Proc. Nat. Acad. Sci. USA 94, 5078–5083) demonstrated a pathway for formation of non-centrosomal microtubules by release from the centrosome. Here we show a parallel pathway not dependent upon the centrosome. Correlative immunostaining with anti-tubulin antibodies and electron microscopy established that apparent free microtubules observed in vivo were not growing ends of long stable microtubules. Free microtubules appeared spontaneously in the cytoplasm and occasionally by breakage of long microtubules. Estimates of the frequencies of free microtubule formation suggest that it can be a relatively common rather than exceptional event in PtK1 cells and may represent a significant source of non-centrosomal microtubules. The observation of free microtubules permitted analysis of the microtubule minus end. Unlike the plus end which showed dynamic instability, the minus end was stable or depolymerized. Breakage of long microtubules generated nascent plus and minus ends; the nascent minus end was generally stable while the plus end was always dynamic. The stability of microtubule minus ends in vivo apparently provides the necessary condition for free microtubule formation in the cytoplasm. Parameters of the dynamic instability of plus ends of free microtubules were similar to those for the distal ends of long microtubules, indicating that the free microtubules were not exceptional in their dynamic behavior. Random walk analysis of microtubule end dynamics gave apparent diffusion coefficients for free and long microtubules which permitted an estimate of turnover half-times. The results support the concept that, in PtK1 cells, a pathway other than plus end dynamics is needed to account for the rapidity of microtubule turnover.

Regulation of Mitosis

1969 ◽  
Vol 5 (3) ◽  
pp. 745-755
Author(s):  
W. T. JACKSON
Keyword(s):  
Mitotic Spindle ◽  
Time Lapse ◽  
Polarization Microscopy ◽  
Animal Cells ◽  
Higher Plant ◽  
Competitive Antagonism ◽  
Dividing Cells ◽  
Spindle Microtubules ◽  
Giant Nuclei

Earlier studies on the effects of the herbicide isopropyl N-phenylcarbamate (IPC) on mitosis revealed blocked metaphases, multinucleate cells, giant nuclei and an increase in number of partly contracted chromosomes. It was assumed that IPC, like colchicine, was causing these effects by disruption of the spindle apparatus by destroying the spindle microtubules. The animal hormone melatonin causes an increase in birefringence of the mitotic spindle in animal cells, presumably by increasing the number of microtubules. We have studied the effects of IPC, melatonin, and combinations of the two on mitosis in dividing endosperm cells of the African blood lily (Haemanthus katherinae Baker) in vivo by phase-contrast and polarization microscopy. Both qualitative and quantitative data are presented. Interpretation of these results has been aided materially by a time-lapse cinemicrographic analysis of dividing cells subjected to 1 and 10 p.p.m. IPC (unpublished) and by an accompanying fine-structural analysis of untreated and IPC-treated cells. Mitosis was disrupted by 0.01-10 p.p.m. IPC, the severity of the effect depending on both concentration and stage of mitosis of the cell at the time of treatment. Concentrations of IPC that caused cessation of chromosome movement also caused loss of birefringence of the mitotic spindle. Melatonin increased birefringence of the mitotic spindle in these plant cells and partly nullified the adverse effects of IPC. The results of this study demonstrate that the herbicide IPC, under our conditions, causes disruption of mitosis and loss of birefringence of the spindle. And it has been established that an animal hormone is capable of increasing the birefringence, and presumably the number of microtubules, of the mitotic spindle in dividing endosperm cells of a higher plant. Although melatonin is capable of partly nullifying the effects of IPC, a competitive antagonism is not postulated.

scholarly journals Reorientation of Mispositioned Spindles in Short Astral Microtubule Mutantspc72Δ Is Dependent on Spindle Pole Body Outer Plaque and Kar3 Motor Protein

2002 ◽  
Vol 13 (4) ◽  
pp. 1366-1380 ◽  
Author(s):  
Dominic Hoepfner ◽  
Florian Schaerer ◽  
Arndt Brachat ◽  
Achim Wach ◽  
Peter Philippsen
Keyword(s):  
Spindle Pole Body ◽  
Time Lapse ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Astral Microtubule ◽  
Spindle Pole Bodies ◽  
Microtubule Motor ◽  
Microtubule Formation ◽  
Mother Cells

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic γ-tubulin complex, can only generate very short (<1 μm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However,SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.

In vivo measurement of apparent diffusion coefficients of hyperpolarized 13 C-labeled metabolites

2014 ◽  
Vol 27 (5) ◽  
pp. 561-569 ◽  
Author(s):  
Lise Vejby Søgaard ◽  
Franz Schilling ◽  
Martin A. Janich ◽  
Marion I. Menzel ◽  
Jan Henrik Ardenkjaer-Larsen
Keyword(s):  
Diffusion Coefficients ◽  
Apparent Diffusion Coefficients ◽  
In Vivo Measurement ◽  
Apparent Diffusion

Measurement of cation fluxes in rat diaphragm

1954 ◽  
Vol 142 (909) ◽  
pp. 497-513 ◽  
Keyword(s):  
Cell Membrane ◽  
Ion Concentration ◽  
Half Time ◽  
Ionic Flux ◽  
Simple Diffusion ◽  
Apparent Diffusion Coefficients ◽  
Significant Difference ◽  
Apparent Diffusion ◽  
The Mean

Calculation of the ionic flux in isolated mammalian muscle at 38° C required a knowledge of internal ion concentration, the cell dimensions, and the kinetics of exchange across the cell membrane. Muscles soaked in Krebs saline showed no indication of fibre swelling or gain of cell water; there was a small fall of intracellular potassium, accompanied by a large rise of sodium. With proper oxygenation, muscle potassium was constant for several hours; anoxia rapidly produced a fall in potassium and gain of sodium. The use of radioactive tracers showed that potassium was completely exchangeable. The mean half-time for exchange of potassium between tissue and saline was 45 min. Initially the rate was somewhat more rapid, but it finally became steady. There was no significant difference between the rates of entry and exit. Potassium exchange was apparently slowed by diffusion through the inter­spaces; the calculated exchange rate across the cell membrane had a half-time of 36 min. The mean potassium flux, after correcting for diffusion, was 21 x 10 -12 equiv. cm -2 s -1 . Fibre sodium exchanged with a half-time of about 10 min, and the outward sodium flux was 28 x 10 -12 equiv. cm -2 s -1 . High values were found for the intercellular space, being 26 ml./100 g in soaked diaphragm muscles as measured by inulin. This was confirmed by a method involving radioactive sodium, and the inulin space in vivo was similar. In their passage through the intercellular fluid, inulin, potassium and sodium appear to follow simple diffusion kinetics, and their apparent diffusion coefficients have been estimated.

scholarly journals Apparent diffusion coefficients of the five major metabolites measured in the human brain in vivo at 3T

2017 ◽  
Vol 79 (6) ◽  
pp. 2896-2901 ◽  
Author(s):  
Dinesh K. Deelchand ◽  
Edward J. Auerbach ◽  
Małgorzata Marjańska
Keyword(s):  
Human Brain ◽  
Diffusion Coefficients ◽  
Apparent Diffusion Coefficients ◽  
Apparent Diffusion

scholarly journals Cytoplasmic dynamics of myosin IIA and IIB: spatial ‘sorting’ of isoforms in locomoting cells

1998 ◽  
Vol 111 (15) ◽  
pp. 2085-2095 ◽  
Author(s):  
J. Kolega
Keyword(s):  
Cultured Cells ◽  
Myosin Ii ◽  
Cell Movement ◽  
Leading Edge ◽  
Time Lapse ◽  
Living Cells ◽  
Immunofluorescence Staining ◽  
Spatial Sorting ◽  
Time Lapse Imaging

Different isoforms of non-muscle myosin II have different distributions in vivo, even within individual cells. In order to understand how these different distributions arise, the distribution and dynamics of non-muscle myosins IIA and myosin IIB were examined in cultured cells using immunofluorescence staining and time-lapse imaging of fluorescent analogs. Cultured bovine aortic endothelia contained both myosins IIA and IIB. Both isoforms distributed along stress fibers, in linear or punctate aggregates within lamellipodia, and diffusely around the nucleus. However, the A isoform was preferentially located toward the leading edge of migrating cells when compared with myosin IIB by double immunofluorescence staining. Conversely, the B isoform was enriched in structures at the cells' trailing edges. When fluorescent analogs of the two isoforms were co-injected into living cells, the injected myosins distributed with the same disparate localizations as endogenous myosins IIA and IIB. This indicated that the ability of the myosins to ‘sort’ within the cytoplasm is intrinsic to the proteins themselves, and not a result of localized synthesis or degradation. Furthermore, time-lapse imaging of injected analogs in living cells revealed differences in the rates at which the two isoforms rearranged during cell movement. The A isoform appeared in newly formed structures more rapidly than the B isoform, and was also lost more rapidly when structures disassembled. These observations suggest that the different localizations of myosins IIA and IIB reflect different rates at which the isoforms transit through assembly, movement and disassembly within the cell. The relative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based structures in that cell.

scholarly journals THE CIRCUMFUSION SYSTEM FOR MULTIPURPOSE CULTURE CHAMBERS

1968 ◽  
Vol 39 (2) ◽  
pp. 430-450 ◽  
Author(s):  
George G. Rose ◽  
M. Kumegawa ◽  
M. Cattoni
Keyword(s):  
Phase Contrast ◽  
Cultured Cells ◽  
Morphological Characteristics ◽  
Time Lapse ◽  
Newborn Mouse ◽  
Contrast Microscopy ◽  
Free Environment ◽  
Parenchymal Cells

The circumfusion system is a complex in vitro pumping unit incorporating 12 multipurpose culture chambers through which a serum-supplemented fluid nutrient is recirculated at a rate of 4.5 ml/min per chamber. This system was used to study the differentiative responses of fetal and newborn mouse liver explants placed in the serum-free environment formed between the sheets of unperforated cellophane and cover glasses of the chambers. Hepatocytes (parenchymal cells) were discernible in 3–5 days. They retained many of their features of differentiation in the circumfusion system for more than 120 days of cultivation. The living morphological characteristics of the hepatocytes were studied by phase-contrast microscopy (direct viewing and time-lapse cinemicrography) and by special cytochemical staining. Electron micrographs were made of both fresh liver specimens and the cultured cells. Comparisons of the cultured parenchymal cells with their in vivo progenitors showed a remarkable preservation of their differentiated state.

scholarly journals Quantitative intravital imaging reveals in vivo dynamics of physiological-stress induced mitophagy

2020 ◽  
Author(s):  
Paul J. Wrighton ◽  
Arkadi Shwartz ◽  
Jin-Mi Heo ◽  
Eleanor D. Quenzer ◽  
Kyle A. LaBella ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Physiological Stress ◽  
Hypoxia Inducible Factor ◽  
Imaging Study ◽  
Time Lapse ◽  
Intravital Imaging ◽  
Response Dynamics ◽  
Pathway Activation

ABSTRACTMitophagy, the selective recycling of mitochondria through autophagy, is a crucial metabolic process induced by cellular stress, and defects are linked to aging, sarcopenia, and neurodegenerative diseases. To therapeutically target mitophagy, the fundamental in vivo dynamics and molecular mechanisms must be fully understood. Here, we generated mitophagy biosensor zebrafish lines expressing mitochondrially targeted, pH-sensitive, fluorescent probes mito-Keima and mito-EGFP-mCherry and used quantitative intravital imaging to illuminate mitophagy during physiological stresses—embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress-response dynamics, and time-lapse imaging revealed mitochondrial filaments undergo piecemeal fragmentation and recycling rather than the wholesale turnover observed in cultured cells. Hypoxia-inducible factor (Hif) pathway activation through physiological hypoxia or chemical or genetic modulation also provoked mitophagy. Intriguingly, mutation of a single mitophagy receptor bnip3 prevented this effect, whereas disruption of other putative hypoxia-associated mitophagy genes bnip3la (nix), fundc1, pink1 or prkn (Parkin) had no effect. This in vivo imaging study establishes fundamental dynamics of fasting-induced mitophagy and identifies bnip3 as the master regulator of Hif-induced mitophagy in vertebrate muscle.

scholarly journals Diffusion-Exchange Weighted Imaging

2009 ◽  
Vol 3 ◽  
pp. MRI.S3504
Author(s):  
Saadallah Ramadan
Keyword(s):  
Pulse Sequence ◽  
Diffusion Method ◽  
Residence Times ◽  
Apparent Diffusion Coefficients ◽  
Apparent Diffusion Coefficient Values ◽  
Apparent Diffusion ◽  
Mean Residence Times ◽  
Signal Dependence ◽  
Cellular Compartments

A method has been developed whereby diffusion and exchange in micro cellular structures in the human brain are correlated to produce a new type of image contrast leading to determination of water exchange rates in vivo. The diffusion method relies on differential apparent diffusion coefficients as detectable nuclei exchange between adjacent compartments marked with different apparent diffusion coefficient values (e.g. intra- and extra-cellular compartments). A new pulse sequence was developed, and used to calculate water intra/extra mean residence times in brain, and the signal dependence on various experimental parameters was analysed. The method was tested in vivo at 3T field strength and produced 160 ms and 550 ms for extra-cellular and intra-cellular mean residence times, respectively.

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