Graduate Thesis Or Dissertation
 

Time-lapse and electron microscope study of cultured epithelium subjected to calcium depletion with EDTA and its subsequent recovery

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  • Primary monolayer cultures of chick renal epithelial cells grown on plastic coverslips were reversibly dissociated with the chelating agent ethylenediaminetetraacetic acid (EDTA). The cellular responses during dissociation and reaggregation in Hank's balanced salt solution (BSS) were observed with phase optics and recorded by time-lapse cinemicrography. The epithelial cells grow as a confluent sheet closely adherent to the plastic substrate. EDTA induces a series of responses by the cells of the monolayer. The chelation of divalent cations by EDTA induces the cells of the monolayer to relinquish mutual adhesive contacts. On newly freed surfaces veiling is initiated for a brief period and then subsides. Cellular contraction continues followed by very active surface blebbing. On continued chelation this activity also ceases at which time the cells are completely separated, fully rounded and highly refractile. Cultures containing fully separated cells were perfused with Hank's BSS and allowed to reassociate into a coherent monolayer. After a brief refractory period surface blebbing resumes and continues for a time giving way to a less vigorous surface expansion. The cells flatten to the substrate, expand centrifugally, and make contact with adjacent cells. This initial contact is extended over greater surface areas until a coherent sheet of cells indistinguishable from the untreated cultures has been established. The EDTA effects are interpreted as being a result of the extraction of divalent cations while the recovery responses are interpreted as a reversal of the EDTA effects by the calcium containing medium. The responses during separation and recovery are discussed in terms of altered sol-gel relationships, reversible cellular permeability properties and reversible changes in the cellular adhesiveness. The sequence of events during separation and reconstitution of the confluent sheet have been studied with the electron microscope to establish changes in the ultrastructure corresponding to those observed with the light microscope. The fine structure of the untreated tissues has been studied with special emphasis on the cellular interrelationships. With the exception of the intracellular cement all adhesive modifications are disrupted during EDTA induced separation and those that are disrupted exhibit a striking recovery during incubation in Hank's BSS. During the separation and recovery sequences microvilli are elaborated at the free surfaces of the cells, they are retracted on fully separated cells and reappear during recovery. Their possible role in cell contact is discussed. In addition to the surface effects EDTA elicits disruption of certain intracellular components. Peripheral microtubules are destroyed during the separation sequence and reappear in the recovery cells, however, those observed in association with the centriole are not affected. The microtubules are discussed in terms of a labile cytoskeleton functioning in formation and maintenance of cell asymmetries. Polyribosomes are decreased in numbers during chelation. This is interpreted as being due to extraction of magnesium by the chelating agent and its removal permits their increase during recovery. The mitochondria, endoplasmic reticulum, Golgi lamellae and the centriole and its derivatives are not altered by treatment. Many of the EDTA induced responses are discussed in the light of events occurring during normal mitosis. The surface responses and cytoplasmic effects of EDTA treatment suggests that a related phenomenon occurs during normal mitosis.
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file details WhiteheadRussellA1967.pdf 2017-08-04 公开 下载