Chapter 11 Use of Cultured Neurons and Neuronal Cell Lines to Study Morphological, Biochemical, and Molecular Changes Occurring in Cell Death

Jc, Mills; Wang, S; Erecińska, Maria; Rn, Pittman

doi:10.1016/s0091-679x(08)61931-7

Cited by 22 publications

(18 citation statements)

References 21 publications

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“…It was also not clear, if they were moving, in which direction they were migrating with respect to the centrosome. To determine these characteristics we used time-lapse video microscopy under special conditions, which allowed prolonged study of cells together with the addition of reagents and washout buffers [Mills et al, 1995]. Figure 6 shows a typical large elongating cell photographed directly from the video screen.…”

Section: Resultsmentioning

confidence: 99%

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Development of polarity in human erythroleukemia cells: Roles of membrane ruffling and the centrosome

Niu

Mills

Nachmias

1997

Cell Motil. Cytoskeleton

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Section: Resultsmentioning

confidence: 99%

“…(The detailed procedure is described in Mills et al [1995].) Important for the present investigation is that the chamber has two holes for tubing; the side tubing carries humidified 5% CO 2 , while the other is for adding medium with appropriate chemicals (e.g., dbcAMP, nocodazole) to the dish.…”

Section: Time-lapse Videomicroscopymentioning

confidence: 99%

Development of polarity in human erythroleukemia cells: Roles of membrane ruffling and the centrosome

Niu

Mills

Nachmias

1997

Cell Motil. Cytoskeleton

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“…These systematic changes were entirely different from those seen in oncotic cardiomyocytes, in which myofibrils were supercontracted and exhibited contraction bands with preserved Z disks. Ca 2ϩ transients appear to be related to the dynamics of apoptosis ubiquitously observed among various cell types (3,9,23,24). These dynamics would be especially apparent in the beating of cells with well-developed myofilament systems, e.g., adult cardiomyocytes, although they would also be reflected in the proteolysis and DNA fragmentation catalyzed by Ca 2ϩ -dependent protease and DNases.…”

Section: Discussionmentioning

confidence: 99%

“…Video recording of various apoptotic cell types in culture has revealed apoptosis to be a dynamic process that is complete within hours and is characterized by several common features: cellular shrinkage and rounding, budding or blebbing, and apoptotic body formation (3,23,24). These structural changes are accompanied by disassembly of the cytoskeletal and nuclear scaffold as a result of proteolysis catalyzed by caspase family proteases (25,36).…”

mentioning

confidence: 99%

Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

Maruyama¹,

Takemura²,

Tohse³

et al. 2006

American Journal of Physiology-Heart and Circulatory Physiology

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2ϩ transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe ␤-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca 2ϩ concentration and cell length. Trains of Ca 2ϩ transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca 2ϩ transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca 2ϩ transients declined and diastolic intracellular Ca 2ϩ concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca 2ϩ channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca 2ϩ -ATPase (thapsigargin), and Na ϩ /Ca 2ϩ exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins (␣-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca 2ϩ transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown. apoptosis; calcium regulation; cardiac myocytes; sarcomere; Z disk SEVERAL REPORTS have suggested that apoptosis among cardiomyocytes plays an important role in the progression of cardiovascular disease (1,7,11). This idea remains controversial, however, because the ultrastructural changes characteristic of apoptosis have only rarely been seen in cardiomyocytes from diseased hearts (8,16,34). However, cardiomyocyte apoptosis can be induced in vitro by a variety of stimuli. For instance, we and others previously described Fas-induced, hypoxia-induced, and ␤-adrenergic pathway-stimulated apoptosis in cultured adult cardiomyocytes (5,15,17,22).Adult cardiomyocytes are terminally differentiated cells that are highly specialized in terms of structure and function. They express contractile proteins and a particularly well-developed cytoskeleton that are tightly packed into the cytoplasm and are sensitive to the Ca 2ϩ load, mediating continual contraction and relaxation in vivo. In an earlier study, we used serial video and electron microscopy to show several unique features of apoptosis in adult cardiomyocytes, including the rhythmic contraction and relaxation (beating) that precede cellular deformation, accelerate as the cell shrinks, and are accompan...

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“…PC12 cells mimic quite closely signal transduction events that occur in sympathetic neurons; in their undifferentiated state they express TrkA and respond to NGF by inducing expression of neuronal proteins and adopting a neuronal morphology (10,11). Prolonged NGF treatment, followed by its withdrawal, induces these cells to undergo programmed cell death closely resembling that seen in sympathetic neurons (12)(13)(14)(15). Undifferentiated PC12 cells also contain synaptic vesicles (16, 17) whose biogenesis and trafficking are controlled by regulated secretory processes similar to those in nerve terminals (18,19).…”

mentioning

confidence: 92%

A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA

Grimes

Beattie²,

Mobley³

1997

Proc. Natl. Acad. Sci. U.S.A.

159

138

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The topology of signal transduction is particularly important for neurons. Neurotrophic factors such as nerve growth factor (NGF) interact with receptors at distal axons and a signal is transduced by retrograde transport to the cell body to ensure survival of the neuron. We have discovered an organelle that may account for the retrograde transport of the neurotrophin signal. This organelle is derived from endocytosis of the receptor tyrosine kinase for NGF, TrkA. In vitro reactions containing semi-intact PC12 cells and ATP were used to enhance recovery of a novel organelle: small vesicles containing internalized NGF bound to activated TrkA. These vesicles were distinct from clathrin coated vesicles, uncoated primary endocytic vesicles, and synaptic vesicles, and resembled transport vesicles in their sedimentation velocity. They contained 10% of the total bound NGF and almost one-third of the total tyrosine phosphorylated TrkA. These small vesicles are compelling candidates for the organelles through which the neurotrophin signal is conveyed down the axon.

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Chapter 11 Use of Cultured Neurons and Neuronal Cell Lines to Study Morphological, Biochemical, and Molecular Changes Occurring in Cell Death

Cited by 22 publications

References 21 publications

Development of polarity in human erythroleukemia cells: Roles of membrane ruffling and the centrosome

Development of polarity in human erythroleukemia cells: Roles of membrane ruffling and the centrosome

Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA

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