Axis establishment and microtubule-mediated waves prior to first cleavage in Beroe ovata

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 75-87 ◽  
Author(s):  
E. Houliston ◽  
D. Carre ◽  
J. A. Johnston ◽  
C. Sardet
Keyword(s):  
Regional Differences ◽  
Cytochalasin B ◽  
Time Lapse ◽  
Zygote Nucleus ◽  
Cell Cycles ◽  
Beroe Ovata ◽  
Minute Period ◽  
Sperm Nuclei ◽  
Reduced Surface ◽  
Peripheral Cytoplasm

The single axis (oral-aboral) and two planes of symmetry of the ctenophore Beroe ovata become established with respect to the position of zygote nucleus formation and the orientation of first cleavage. Bisection of Beroe eggs at different times revealed that differences in egg organisation are established in relation to the presumptive oral-aboral axis before first cleavage. Lateral fragments produced after but not before the time of first mitosis developed into larvae lacking comb-plates on one side. Time-lapse video demonstrated that waves of cytoplasmic reorganisation spread through the layer of peripheral cytoplasm (ectoplasm) of the egg during the 80 minute period between pronuclear fusion and first cleavage, along the future oral-aboral axis. These waves are manifest as the progressive displacement and dispersal of plaques of accumulated organelles around supernumerary sperm nuclei, and a series of surface movements. Their timing and direction of propagation suggest they may be involved in establishing cytoplasmic differences with respect to the embryonic axis. Inhibitor experiments suggested that the observed cytoplasmic reorganisation involves microtubules. Nocodazole and taxol, which prevent microtubule turnover, blocked plaque dispersal and reduced surface movements. The microfilament-disrupting drug cytochalasin B did not prevent plaque dispersal but induced abnormal surface contractions. We examined changes in microtubule organisation using immunofluorescence on eggs fixed at different times and in live eggs following injection of rhodamine-tubulin. Giant microtubule asters become associated with each male pronucleus after the end of meiosis. Following pronuclear fusion they disappear successively, those nearest the zygote nucleus shrinking first, to establish gradients of aster size within single eggs. Regional differences in microtubule behaviour around the time of mitosis were revealed by brief taxol treatment, which induced the formation of small microtubule asters in the region of the nucleus or spindle during both first and second cell cycles. The observed wave of change may thus reflect the local appearance and spreading of mitotic activity as the zygote nucleus approaches mitosis.

scholarly journals Motility of bile canaliculi in the living animal: implications for bile flow.

1991 ◽  
Vol 113 (5) ◽  
pp. 1069-1080 ◽  
Author(s):  
N Watanabe ◽  
N Tsukada ◽  
C R Smith ◽  
M J Phillips
Keyword(s):  
Bile Flow ◽  
Cytochalasin B ◽  
Time Lapse ◽  
Sodium Fluorescein ◽  
Branch Points ◽  
Bile Canaliculi ◽  
Enhanced Fluorescence ◽  
Microscopic Techniques

Modern fluorescence microscopic techniques were used to image the bile canalicular system in the intact rat liver, in vivo. By combining the use of sodium fluorescein secretion into bile, with digitally enhanced fluorescence microscopy and time-lapse video, it was possible to capture and record the canalicular motility events that accompany the secretion of bile in life. Active bile canalicular contractions were found predominantly in zone 1 (periportal) hepatocytes of the liver. The contractile movements were repetitive, forceful, and appeared unidirectional moving bile in a direction towards the portal bile ducts. Contractions were not seen in the network of canaliculi on the surface of the liver. Cytochalasin B administration resulted in reduced canalicular motility, progressive dilation of zone 1 canaliculi, and impairment of bile flow. Canalicular dilations invariably involved the branch points of the canalicular network. The findings add substantively to previous in vitro studies using couplets, and suggest that canalicular contractions contribute physiologically to bile flow in the liver.

scholarly journals Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization.

1991 ◽  
Vol 114 (5) ◽  
pp. 929-940 ◽  
Author(s):  
M Terasaki ◽  
L A Jaffe
Keyword(s):  
Sea Urchin ◽  
Time Lapse ◽  
Sperm Nucleus ◽  
Temporal Organization ◽  
Calcium Wave ◽  
Zygote Nucleus ◽  
Lytechinus Pictus ◽  
Sea Urchin Egg ◽  
A Cell ◽  
Sperm Aster

The ER of eggs of the sea urchin Lytechinus pictus was stained by microinjecting a saturated solution of the fluorescent dicarbocyanine DiIC18(3) (DiI) in soybean oil; the dye spread from the oil drop into ER membranes throughout the egg but not into other organelles. Confocal microscopy revealed large cisternae extending throughout the interior of the egg and a tubular membrane network at the cortex. Since diffusion of DiI is confined to continuous bilayers, the spread of the dye supports the concept that the ER is a cell-wide, interconnected compartment. In time lapse observations, the internal cisternae were seen to be in continuous motion, while the cortical ER was stationary. After fertilization, the internal ER appeared to become more finely divided, beginning as a wave apparently coincident with the calcium wave and becoming most marked by 2-3 min. By 5-8 min the ER returned to an organization similar to that of the unfertilized egg. The cortical network also changed at fertilization; it became disrupted and eventually recovered. DiI labeling allowed continuous observations of the ER during pronuclear migration and mitosis. DiI-stained membranes accumulated in the region of the microtubule array surrounding the sperm nucleus and centriole (the sperm aster) as it migrated to the center of the egg; this accumulation persisted near the centrosomes and zygote nucleus throughout pronuclear fusion and the first two mitotic cycles. We have used a new method to observe the spatial and temporal organization of the ER in a living cell, and we have demonstrated a striking reorganization of the ER at fertilization.

scholarly journals Blood Platelets Are Assembled Principally at the Ends of Proplatelet Processes Produced by Differentiated Megakaryocytes

1999 ◽  
Vol 147 (6) ◽  
pp. 1299-1312 ◽  
Author(s):  
Joseph E. Italiano ◽  
Patrick Lecine ◽  
Ramesh A. Shivdasani ◽  
John H. Hartwig
Keyword(s):  
Cytochalasin B ◽  
De Novo ◽  
Blood Platelets ◽  
Time Lapse ◽  
Video Microscopy ◽  
Platelet Production ◽  
Time Lapse Microscopy ◽  
Complex Process ◽  
Dynamic Structures

Megakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the megakaryocyte cytoplasm. We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.

scholarly journals Growth of neurites without filopodial or lamellipodial activity in the presence of cytochalasin B.

1984 ◽  
Vol 99 (6) ◽  
pp. 2041-2047 ◽  
Author(s):  
L Marsh ◽  
P C Letourneau
Keyword(s):  
Cytochalasin B ◽  
Ganglion Cells ◽  
Video Recording ◽  
Neurite Growth ◽  
Time Lapse ◽  
Growth Cones ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Actin Networks ◽  
Dorsal Root Ganglion Cells

To examine the role in neurite growth of actin-mediated tensions within growth cones, we cultured chick embryo dorsal root ganglion cells on various substrata in the presence of cytochalasin B. Time-lapse video recording was used to monitor behaviors of living cells, and cytoskeletal arrangements in neurites were assessed via immunofluorescence and electron microscopic observations of thin sections and whole, detergent-extracted cells decorated with the S1 fragment of myosin. On highly adhesive substrata, nerve cells were observed to extend numerous (though peculiarly oriented) neurites in the presence of cytochalasin, despite their lack of both filopodia and lamellipodia or the orderly actin networks characteristic of typical growth cones. We concluded that growth cone activity is not necessary for neurite elongation, although actin arrays seem important in mediating characteristics of substratum selectivity and neurite shape.

scholarly journals Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development

2000 ◽  
Vol 11 (3) ◽  
pp. 897-914 ◽  
Author(s):  
Mark Terasaki
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Permeability Barrier ◽  
Cell Cycles ◽  
Nuclear Envelope Breakdown ◽  
Quantitative Measurements ◽  
Green Fluorescent

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2–3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is ∼30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-μm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.

Regional differences in the morphology and motility of mesodermal cells from the early wing-bud of normal and talpid3 mutant chick embryos

Development ◽  
1978 ◽  
Vol 48 (1) ◽  
pp. 185-203
Author(s):  
D. A. Bell ◽  
D. A. Ede
Keyword(s):  
Regional Differences ◽  
Time Lapse ◽  
Distal Region ◽  
Limb Bud ◽  
Scanning Electron Micrographs ◽  
Normal Cells ◽  
Wing Bud ◽  
Scanning Electron

A method of culturing has been employed to compare the properties of cells migrating from small mesodermal explants taken from different regions of normal and mutant limb-buds at different stages of development. An analysis by time-lapse cinematography of the morphology and mobility of cells migrating from explants defines a distal region within the limb-bud where these properties are distinct from those of cells from more proximal regions. In the normal wing-bud distal cells subjacent to the apical ectodermal ridge possess a characteristic multipolar morphology and translocate slowly in vitro. Cells from more proximal regions tend to be bipolar and translocate more rapidly. Distal and proximal cells also probably differ in their adhesive strengths. In the mutant, talpid3, distal and proximal cells do not differ in the above properties and cells from all regions of the limb-bud are multipolar, translocate slowly and are more adhesive than normal cells. A study of light micrographs and scanning electron micrographs suggests that these regional differences are found in the limb-bud in vivo and are not merely an effect produced by the in vitro culturing system.

Microtubule distribution and function in early Pelvetia development

1990 ◽  
Vol 97 (3) ◽  
pp. 545-552
Author(s):  
DARRYL L. KROFF ◽  
ANNA MADDOCK ◽  
DAVID L. GARD
Keyword(s):  
Mitotic Spindle ◽  
Immunofluorescence Microscopy ◽  
Cell Lineage ◽  
Cytochalasin D ◽  
Division Plane ◽  
Cell Cycles ◽  
Growth Axis ◽  
And Function ◽  
Proper Orientation ◽  
Peripheral Cytoplasm

We have used immunofluorescence microscopy to examine the distribution of microtubules (Mts) during the first two cell cycles in embryos of the brown alga, Pelvetia. Prior to germination of the zygote at 12 h post-fertilization, Mts radiated from the circumnuclear region into the peripheral cytoplasm. After rhizoid emergence, Mts resolved into two perinuclear microtubule organizing centers (MTOCs). The axis defined by the pair of MTOCs was oriented transverse to the growth axis, with Mts extending from each MTOC into the rhizoid. The axis defined by the MTOCs then reoriented by 90 degrees, and aligned with the growth axis. The first mitotic spindle formed between these MTOCs. The division plane bisected the spindle, giving rise to rhizoid and thallus cells with distinct developmental potentials. During the second cell cycle, the axis defined by MTOCs in the rhizoid cell again reoriented from an orthogonal to an axial alignment with respect to the growth axis. MTOC reorientation did not occur in the thallus cell, and the division planes in the rhizoid and thallus cells were orthogonal to one another. Zygotes treated with amiprophos methyl (APM) or taxol established an axis and initiated rhizoid outgrowth. However, treated zygotes ceased growing soon after germination and failed to divide. Cytochalasin D, which prevents establishment of the developmental axis, interfered with the proper orientation of the spindle. From these results we conclude that: (1) Mts are not required for establishment of the rhizoid-thallus axis or rhizoid germination; (2) an F-actin-dependent process, probably establishment of a developmental axis, is required for rotation of the axis defined by MTOCs; and (3) the alignment of perinuclear MTOCs dictates the orientation of spindle and subsequent division planes, and thereby controls cell lineage.

Cell development in the anther, the ovule, and the young seed of Triticum aestivum L. var. Chinese Spring

1973 ◽  
Vol 266 (875) ◽  
pp. 39-81 ◽  
Keyword(s):  
Plant Species ◽  
Chinese Spring ◽  
Embryo Sac ◽  
Cell Development ◽  
Anther Dehiscence ◽  
Higher Plant ◽  
Cell Cycles ◽  
Wide Range ◽  
Sperm Nuclei ◽  
Antipodal Cells

The developmental behaviour of reproductive cells was studied during premeiotic mitotic activity, premeiotic interphase, meiosis in anthers and ovaries, microsporogenesis, megasporogenesis and embryo and endosperm growth in Triticum aestivum L. var. Chinese Spring. Particular attention was paid first to the timing and rate of cell development in the anthers and the ovary within a floret and secondly , to the timing and rate of nuclear and cell development in the young embryo and endosperm. At 20 °C the development studied lasted in each floret about 21 days starting 7 days prior to meiosis in anthers and ending 5 days after anther dehiscence and pollination. The durations of up to twenty successive cell cycles were estimated. In anthers of plants grown at 20 °C the durations of the three successive cell cycles immediately prior to the cycle which ends at first anaphase of meiosis were about 25, 35 and 55 h respectively. The increase in cell cycle time was correlated with an increase in the size of archesporial cells and their nuclei. A progressive increase in the durations of successive cell cycles as meiosis is approached has not been measured previously in a higher plant species, although it has been noted in the germ line cells of male mice. The pollen mother cells (p.m.cs) within an anther were synchronized prior to meiosis by having their development blocked somewhere in G 1 of premeiotic interphase. The developmental hold began to operate about 103 h prior to the synchronous onset of meiosis in all the p.m.cs within an anther at 20 °C. About 55 h later, when the last archesporial cell completed its final premeiotic mitosis, all the p.m.cs had accumulated in G 1 of premeiotic interphase and synchrony was complete. Premeiotic interphase after all the p.m.cs were first synchronized in G 1 until the synchronous onset of meiosis lasted about 48 h. During this period the G 1 developmental hold was released and p.m.cs initiated DNA synthesis synchronously about 12 to 15 h prior to the start of leptotene. Meiosis in p.m.cs lasted 24 h at 20 °C. Within each floret, meiosis in p.m.cs was almost or quite synchronous with, and had the same duration as, meiosis in the embryo sac mother cell. The Q 10 for meiosis in p.m.cs over the temperature range 15 to 25 °C was about 2.3. Microsporogenesis from tetrad stage until anther dehiscence lasted about 7.5 days at 20 °C. The first pollen grain mitosis (p.g.m. 1) occurred 2.5 days and second pollen grain mitosis (p.g.m. 2) 5.0 days after the end of meiosis. Concurrent with p.g.m. 1 the functional megaspore in the ovule of the same floret divided. This division was rapidly followed by two more synchronous division cycles (also concurrent with p.g.m. 1) which produced an 8-nucleate embryo sac. By p.g.m. 2 the embryo sac contained 20 to 30 antipodal cells at its chalazal end. The antipodal cells subsequently became highly polyploid and some eventually contained up to 200 times as much DNA as haploid egg nuclei. At 20 °C the sperm nuclei reached the egg and polar nuclei about 40 min after pollination. The primary endosperm nucleus divided about 6 h after pollination while the zygote did not divide until about 22 h after pollination. The endosperm often contained 16 mitotic nuclei 24 h after pollination. The nuclear division cycle during the first five division cycles was about 4.5 h. Until the tenth division cycle when the endosperm became a cellular tissue, development of endosperm nuclei was synchronous, but thereafter synchrony was progressively lost. Early embryo development was marked by a gradual decrease in the durations of successive cell cycles. This decrease was apparently correlated with a decrease in the size of embryo cells and their nuclei. Nuclear and cellular developmental rates at 20 °C were very variable. Estimates of nuclear cycle times ranged from about 60 h in the microspore to about 4.5 h in some endosperm nuclei. Nuclear volume was also very variable and ranged from about 240 μm 3 for sperm nuclei in mature microspores to about 160000 μm3 in some polyploid antipodal cells. Both the wide range of nuclear types described, and the speed with which nuclear characters changed, illustrates the remarkable plasticity of the wheat nucleus which may occur in several very different forms. A comprehensive and integrated study of development at the cellular level in reproductive tissues of a higher plant species is presented. The importance of this study is twofold. First, it allows the comparison of reproductive cell behaviour in a higher plant species and in those animal species which have been intensively examined. Secondly the availability of a description of ‘normal’ development under controlled conditions in euploid plants of Chinese Spring opens the way for comparative studies using the wide range of available mutant or chromosomally different genotypes of Chinese Spring which are known to vary in their reproductive development.

The structure of the human cell cycle

2021 ◽  
Author(s):  
Wayne Stallaert ◽  
Katarzyna M. Kedziora ◽  
Colin D. Taylor ◽  
Tarek M. Zikry ◽  
Holly K. Sobon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Human Cell ◽  
Time Lapse ◽  
Underlying Structure ◽  
Cell Cycle Exit ◽  
Multiple Points ◽  
Cell Cycles ◽  
Single Cell Imaging ◽  
Proliferative Cell

ABSTRACTThe human cell cycle is conventionally depicted as a five-phase model consisting of four proliferative phases (G1, S, G2, M) and a single state of arrest (G0). However, recent studies show that individual cells can take different paths through the cell cycle and exit into distinct arrest states, thus necessitating an update to the canonical model. We combined time lapse microscopy, highly multiplexed single cell imaging and manifold learning to determine the underlying “structure” of the human cell cycle under multiple growth and arrest conditions. By visualizing the cell cycle as a complete biological process, we identified multiple points of divergence from the proliferative cell cycle into distinct states of arrest, revealing multiple mechanisms of cell cycle exit and re-entry and the molecular routes to senescence, endoreduplication and polyploidy. These findings enable the visualization and comparison of alternative cell cycles in development and disease.One-sentence summaryA systems-level view of single-cell states reveals the underlying architecture of the human cell cycle

scholarly journals Time-Lapse Video Microscopy Analysis Reveals Astral Microtubule Detachment in the Yeast Spindle Pole Mutantcnm67

2000 ◽  
Vol 11 (4) ◽  
pp. 1197-1211 ◽  
Author(s):  
Dominic Hoepfner ◽  
Arndt Brachat ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Attachment Site ◽  
Time Lapse ◽  
Spindle Pole ◽  
Cell Cycles ◽  
Astral Microtubule ◽  
Normal Behavior ◽  
Green Fluorescent ◽  
Diploid Cells

Saccharomyces cerevisiae cnm67Δ cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein–labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the γ-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Δ cells Spc72–γ-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Δ cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.

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